APC Antibodies

ABSTRACT

A human gene termed APC is disclosed. Methods and kits are provided for assessing mutations of the APC gene in human tissues and body samples. APC mutations are found in familial adenomatous polyposis patients as well as in sporadic colorectal cancer patients. APC is expressed in most normal tissues. These results suggest that APC is a tumor suppressor.

This application is a division, of application Ser. No. 08/289,548,filed Aug. 12, 1994, which is a division of application Ser. No.07/741,940 filed Aug. 8, 1991 (issued as U.S. Pat. No. 5,352,775).

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of grantsawarded by the National Institutes or Health.

TECHNICAL AREA OF THE INVENTION

The invention relates to the area of cancer diagnostics andtherapeutics. More particularly, the invention relates to detection ofthe germline and somatic alterations of wild-type APC genes. Inaddition, it relates to therapeutic intervention to restore the functionof APC gene product.

BACKGROUND OF THE INVENTION

According to the model of Knudson for tumorigenesis (Cancer Research,Vol. 45, p. 1482, 1985), there are tumor suppressor genes in all normalcells which, when they become non-functional due to mutation, causeneoplastic development. Evidence for this model has been found in thecases of retinoblastoma and colorectal tumors. The implicated suppressorgenes in those tumors, RB, p53, DCC and MCC, were found to be deleted oraltered in many cases of the tumors studied. (Hansen and Cavenee, CancerResearch, Vol. 47, pp: 5518-5527 (1987); Baker et al., Science, Vol.244, p. 217 (1989); Fearon et al., Science, Vol. 247, p. 49 (1990);Kinzler et al. Science Vol. 251. p. 1366 (1991).)

In order to fully understand the pathogenesis of tumors, it will benecessary to identify the other suppressor genes that play a role in thetumorigenesis process. Prominent among there is the one(s) presumptivelylocated at 5q21. Cytogenetic (Herrera et al., Am. J. Med. Genet., Vol.25, p. 473 (1986) and linkage (Leppert et al., Science, Vol. 238, p.1411 (1987); Bodmer et al., Nature, Vol. 328, p. 614 (1987)) studieshave shown that this chromosome region harbors the gene responsible forfamilial adenomatous polyposis (FAP) and Gardner's Syndrome (GS). FAP isan autosomal-dominant, inherited disease in which affected individualsdevelop hundreds to thousands of adenomatous polyps, some of whichprogress to malignancy. GS is a variant of FAP in which desmold tumors,osteomas and other soft tissue tumors occur together with multipleadenomas of the colon and rectum. A less severe form of polyposis hasbeen identified in which only a few (2-40) polyps develop. Thiscondition also is familial and is linked to the same chromosomal markersas FAP and GS (Leppert et al., New England Journal of Medicine, Vol.322, pp. 904-908, 1990.) Additionally, this chromosomal region is oftendeleted from the adenomas (Vogelstein et al., N. Engl. J. Med., Vol.319, p. 525 (1988)) and carcinomas (Vogelstein et al., N. Engl. J. Med.,Vol. 319, p. 525 (1988); Solomon et al., Nature, Vol. 328, p. 616(1987); Sasaki et al., Cancer Research, Vol. 49, p. 4402 (1989);Delattre et al., Lancet, Vol. 2, p. 353 (1989); and Ashton-Rickardt etal., Oncogene, Vol. 4, p. 1169 (1989)) of patients without FAP (sporadictumors). Thus, a putative suppressor gene on chromosome 5q21 appears toplay a role in the early stages of colorectal neoplasia in beth bothsporadic and familial tumors.

Although the MCC gene has been identified on 5q21 as a candidatesuppressor gene, it does not appear to be altered in FAP or GS patients.Thus there is a need in the art for investigations of this chromosomalregion to identify genes and to determine if any of such genes areassociated with FAP and/or GS and the process of tumorigenesis.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method fordiagnosing and prognosing a neoplastic tissue of a human.

It is another object of the invention to provide a method of detectinggenetic predisposition to cancer.

It is another object of the invention to provide a method of supplyingwild-type APC gene function to a cell which has lest said gene function.

It is yet another object of the invention to provide a kit fordetermination of the nucleotide sequence of APC alleles by thepolymerase chain reaction.

It is still another object of the invention to provide nucleic acidprobes for detection of mutations in the human APC gene.

It is still another object of the invention to provide a cDNA moleculeencoding the APC gene product.

It is yet another object of the invention to provide a preparation ofthe human APC protein.

It is another object of the invention to provide a method of screeningfor genetic prodisposition predisposition to cancer.

It is an object of the invention to provide methods of testingtherapeutic agents for the ability to suppress neoplasia.

It is still another object of the invention to provide animals carryingmutant APC alleles.

These and other objects of the invention are provided by one or more ofthe embodiments which are described below. In one embodiment of thepresent invention a method of diagnosing or prognosing a neoplastictissue of a human is provided comprising: detecting somatic alterationof wild-type APC genes or their expression products in a sporadiccolorectal cancer tissue, said alteration indicating neoplasia of thetissue.

In yet another embodiment a method is provided of detecting geneticpredisposition to cancer in a human including familial adenomatouspolyposis (FAP) and Gardner's Syndrome (GS), comprising: isolating ahuman sample selected from the group consisting of blood and fetaltissue; detecting alteration of wild-type APC gene coding sequences ortheir expression products from the sample, said alteration indicatinggenetic predisposition to cancer.

In another embodiment of the present invention a method is provided forsupplying wild-type APC gene function to a cell which has lost said genefunction by virtue of a mutation in the APC gene, comprising:introducing a wild-type APC gene into a cell which has lost said genefunction such that said wild-type gene is expressed in the cell.

In another embodiment a method of supplying wild-type APC gene functionto a cell is provided comprising: introducing a portion of a wild-typeAPC gene into a cell which has lost said gene function such that saidportion is expressed in the cell, said portion encoding a part of theAPC protein which is required for non-neoplastic growth of said cell.APC protein can also be applied to cells or administered to animals toremediate for mutant APC genes. Synthetic peptides or drugs can also beused to mimic APC function in cells which have altered APC expression.

In yet another embodiment a pair of single stranded primers is providefor determination of the nucleotide sequence of the APC gene bypolymerase chain reaction. The sequence of said pair of single strandedDNA primers is derived from chromosome 5q band 21, said pair of primersallowing synthesis of APC gene coding sequences.

In still another embodiment of the invention a nucleic acid probe isprovided which is complementary to human wild-type APC gene cedingsequences and which can form mismatches with mutant APC genes, therebyallowing their detection by enzymatic or chemical cleavage or by shiftsin electrophoretic mobility.

In another embodiment of the invention a method is provided fordetecting the presence of a neoplastic tissue in a human. The methodcomprises isolating a body sample from a human; detecting in said samplealteration of a wild-type APC gene sequence or wild-type APC expressionproduct, said alteration indicating the presence of a neoplastic tissuein the human.

In still another embodiment a cDNA molecule is provided which comprisesthe coding sequence of the APC gene.

In even another embodiment a preparation of the human APC protein isprovided which is substantially free of other human proteins. The aminoacid sequence of the protein is shown in FIG. 3 FIGS. 3A-3Z (SEQ ID NOS:7 and 2).

In yet another embodiment of the invention a method is provided forscreening for genetic predisposition to cancer, including familialadenomatous polyposis (FAP) and Gardner's Syndrome (GS), in a human. Themethod comprises: detecting among kindered persons the presence of a DNApolymorphism which is linked to a mutant APC allele in an individualhaving a genetic predisposition to cancer, said kindered beinggenetically related to the individual, the presence of said polymorphismsuggesting a predisposition to cancer.

In another embodiment of the invention a method of testing therapeuticagents for the ability to suppress a neoplastically transformedphenotype is provided. The method comprises: applying a test substanceto a cultured epithelial cell which carries a mutation in an APC allele;and determining whether said test substance suppresses theneoplastically transformed phenotype of the cell.

In another embodiment of the invention a method of testing therapeuticagents for the ability to suppress a neoplastically transformedphenotype is provided. The method comprises: administering a testsubstance to an animal which carries a mutant APC allele; anddetermining whether said test substance prevents or suppresses thegrowth of tumors.

In still other embodiments of the invention transgenic animals areprovided. The animals carry a mutant APC allele from a second animalspecies or have been genetically engineered to contain an insertionmutatation which disrupts an APC allele.

The present invention provides the art with the information that the APCgene, a heretofore unknown gene is, in fact, a target of mutationalalterations on chromosome 5q21 and that these alterations are associatedwith the process of tumorigenesis. This information allows highlyspecific assays to be performed to assess the neoplastic status of aparticular tissue or the predisposition to cancer of an individual. Thisinvention has applicability to Familial Adenomatous Polyposis, sporadiccolorectal cancers, Guardner's Syndrome, as well as the less severefamilial polyposis discusses above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an overview of yeast artificial chromosome (YAC) contigs.Genetic distances between selected RFLP markers from within the contigsare shown in centi-Morgans.

FIGS. 1B-1, 1B-2 and 1B-3 show a detailed map of the three centralcontigs. The position of the six identified genes from within the FAPregion is shown: the 5′ and 3′ ends of the transcripts from these geneshave in general not yet been isolated, as indicated by the string ofdots surrounding the bars denoting the genes' positions. Selectedrestriction endonuclease recognition sites are indicated. B, BssH2; S,SstII; M, MluI; N, NruI.

FIGS. 2A and 2B show the sequence of TB1 (FIG. 2A, SEQ ID NO:5) and TB2(FIG. 2B, SEQ ID NO:6) genesproteins. The cDNA sequence of the TB1 genewas determined from the analysis of 11 cDNA clones derived from normalcolon and liver, as described in the text. A total of 2314 bp werecontained within the overlapping cDNA clones, defining an ORF of 424amino acids beginning at nucleotide 1. Only the predicted amino acidsfrom the ORF are shown. The carboxy-terminal end of the ORF hasapparently been identified, but the 5′ end of the TB1 transcript has notyet been precisely determined.

The cDNA sequence of the TB2 gene was determined from the YS-39 clonederived as described in the text. This clone consisted of 2300 bp anddefined an ORF of 185 amino acids beginning at nucleotide 1. Only thepredicted amino acids are shown. The carboxy terminal end of the ORF hasapparently been identified, but the 5′ end of the TB2 transcript has notbeen precisely determined.

FIGS. 3A-3F FIGS. 3A-3Zshow the sequence of the APC gene product (SEQ IDNO:7). The cDNA sequence was determined through the analysis of 87 cDNAclones derived from normal colon, liver, and brain. A total of 8973 bpwere contained within overlapping cDNA clones, defining an ORF of28422843 amino acids. In frame stop codons surrounded this ORF, asdescribed in the text, suggesting that the entire APC gene product wasrepresented in the ORF illustrated. Only the predicted amino acids areshown.

FIGS. 4A and 4B show the local similarity between human APC (SEQ IDNO:2) and ral2 (SEQ ID NO:8) of yeast. FIG. 4A shows amino acids 203 to233 of APC, and FIG. 4B shows amino acids 453 to 481 of APC. Localsimilarity among the APC (SEQ ID NO:2) and MCC genes (SEQ ID NO:10)genes and the m3 muscarinic acetylcholine receptor (SEQ ID NO:9) isshown. The region of the mAChR shown corresponds to that responsible forcoupling the receptor to G proteins. The connecting lines indicateidentities; dots indicate related amino acids residues.

FIG. 5 shows the genomic map of the 1200 kb NotI fragment at the FAPlocus. The NotI fragment is shown as a bold line. Relevant parts of thedeletion chromosomes from patients 3214 and 3824 are shown as stippledlines. Probes used to characterize the NotI fragment and the deletions,and three YACs from which subclones were obtained, are shown below therestriction map. The chimeric end of YAC 183H12 is indicated by a dottedline. The orientation and approximate position of MCC are identifiedabove the map.

FIG. 6A-6D show the DNA sequence (SEQ ID NO:3) and predicted amino acidsequence of DP1 (TB2) (SEQ ID NO:4). The nucleotide numbering begins atthe most 5′ nucleoitde isolated. A proposed initiation methionine (base77) is indicated in bold type. The entire coding sequence is presented.

FIG. 7A, FIG. 7B-1, and FIG. 7B-2 show the arrangement of exons in DP2.5(APC). (A) Exon 9 corresponds to nucleotides 933-1312; exon 9acorresponds to nucleotides 1236-1312. The stop codon in the cDNA is atnucleotide 8535. (B) Partial intronic sequence surrounding each exon isshown (SEQ ID NO: 11-38). 5′ intron sequences of exons 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, and 15 are shown in SEQ ID NOS: 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, respectively. 3′ intronsequences of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 areshown in SEQ ID NOS: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, respectively.

DETAILED DESCRIPTION

It is a discovery of the present invention that mutational eventsassociated with tumorigenesis occur in a previously unknown gene onchromosome 5q named here the APC (Adenomatous Polyposis Coil) gene.Although it was previously known that deletion of alleles of chromosome5q were common in certain types of cancers, it was not known that atarget gene of these deletions was the APC gene. Further it was notknown that other types of mutational events in the APC gene and alsoassociated with cancers. The mutations of the APC gene can involve grossrearrangements, such as insertions and deletions. Point mutations havealso been observed.

According to the diagnostic and prognostic method of the presentinvention, alteration of the wild-type APC gene is detected. “Alterationof a wild-type gene” according to the present invention encompasses allforms of mutations—including deletions. The alteration may be due toeither rearrangements such as insertions, inversions, and deletions, orto point mutations. Deletions may be of the entire gene or only aportion of the gene. Somatic mutations are those which occur only incertain tissues, e.g., in the tumor tissue, and are not inherited in thegermline. Germline mutations can be found in any of a body's tissues. Ifonly a single allele is somatically mutated, an early neoplastic stateis indicated. However, if both alleles are mutated then a lateneoplastic state is indicated. The finding of APC mutations thusprovides both diagnostic and prognostic information. An APC allele whichis not deleted (e.g., that on the sister chromosome to a chromosomecarrying an APC deletion) can be screened for other mutations, such asinsertions, small deletions, and point mutations. It is believed thatmany mutations found in tumor tissues will be those leading to decreasedexpression of the APC gene product. However, mutations leading tonon-functional gene products would also lead to a cancerous state. Pointmutational events may occur in regulatory regions, such as in thepromoter of the gene, leading to loss or diminution of expression of themRNA. Point mutations may also abolish proper RNA processing, leading toloss of expression of the APC gene product.

In order to detect the alteration of the wild-type APC gene in a tissue,it is helpful to isolate the tissue free from surrounding normaltissues. Means for enriching a tissue preparation for tumor cells areknown in the art. For example, the tissue may be isolated from paraffinor cryostat sections. Cancer cells may also be separated from normalcells by flow cytometry. These as well as other techniques forseparating tumor from normal cells are well known in the art. If thetumor tissue is highly contaminated with normal cells, detection ofmutations is more difficult.

Detection of point mutations may be accomplished by molecular cloning ofthe APC allele (or alleles) and sequencing that allele(s) usingtechniques well known in the art. Alternatively, the polymerase chainreaction (PCR) can be used to amplify gene sequences directly from agenomic DAN preparation from the tumor tissue. The DNA sequence of theamplified sequences can then be determined. The polymerase chainreaction itself is well known in the art. See, e.g., Saiki et al.,Science, Vol. 239, p. 487, 1988; U.S. Pat. No. 4,683,203; and U.S. Pat.No. 4,683,195. Specific primers which can be used in order to amplifythe gene will be discussed in more detail below. The ligase chainreaction, which is known in the art, can also be used to amplify APCsequences. See Wu et al., Genomics, Vol. 4, pp. 560-569 (1989). Inaddition, a technique known as allele specific PCR can be used. (SeeRuano and Kidd, Nucleic Acids Research, Vol. 17, p. 8392, 1989.)According to this technique, primers are used which hybridize at their3′ ends to a particular APC mutation. If the particular APC mutation isnot present, an amplification product is not observed. AmplificationRefractory Mutation System (ARMS) can also be used as disclosed inEuropean Patent Application Publication No. 0332435 and in Newton etal., Nucleic Acids Research, Vol. 17, p.7, 1989. Insertions anddeletions of genes can also be detected by cloning, sequencing andamplification. In addition, restriction fragment length polymorphism(RFLP) probes for the gene or surrounding marker genes can be used toscore alteration of an allele or an insertion in a polymorphic fragment.Such a method is particularly useful for screening among kinderedpersons of an affected individual for the presence of an APC mutationfound in that individual. Single stranded conformation polymorphism(SSCP) analysis can also be used to detect base change variants of anallele. (Orita et al., Proc. Natl. Acad. Sci. USA Vol. 86, pp.2766-2770, 1989, and Genomics, Vol. 5, pp. 874-879, 1989.) Othertechniques for detecting insertions and deletions as are known in theart can be used.

Alteration of wild-type genes can also be detected on the basis of thealteration of a wild-type expression product of the gene. Suchexpression products include both the APC mRNA as well as the APC proteinproduct. The sequences of these products are shown in FIG. 3 FIGS.3A-3Z. Point mutations may be detected by amplifying and sequencing themRNA or via molecular cloning of cDNA made from the mRNA. The sequenceof the cloned cDNA can be determined using DNA sequencing techniqueswhich are well known in the art. The cDNA can also be sequenced via thepolymerase chain reaction (PCR) which will be discussed in more detailbelow.

Mismatches, according to the present invention are hybridized nucleicacid duplexes which are not 100% homologous. The lack of total homologymay be due to deletions, insertions, inversions, substitutions orframeshift mutations. Mismatch detection can be used to detect pointmutations in the gene or its mRNA product. While these techniques areless sensitive than sequencing, they are simpler to perform on a largenumber of tumor samples. An example of a mismatch cleavage technique isthe RNase protection method, which is described in detail in Winter etal., Proc. Nat. Acad. Sci. USA, Vol. 82, p. 7575, 1985 and Meyers etal., Science, Vol. 230, p. 1242, 1985. In the practice of the presentinvention the method involves the use of a labeled riboprobe which iscomplementary to the human wild-type APC gene coding sequence. Theriboprobe and either mRNA or DNA isolated from the tumor tissue areannealed (hybridized) together and subsequently digested with the enzymeRNase A which is able to detect some mismatches in a duplex RNAstructure. If a mismatch is detected by RNase A, it cleaves at the siteof the mismatch. Thus, when the annealed RNA preparation is separated onan electrophoretic gel matrix, if a mismatch has been detected andcleaved by RNase A, an RNA product will be seen which is smaller thanthe full-length duplex RNA for the riboprobe and the mRNA or DNA. Theriboprobe need not be the full length of the APC mRNA or gene but can bea segment of either. II If the riboprobe comprises only a segment of theAPC mRNA or gene it will be desirable to use a number of these probes toscreen the whole mRNA sequence for mismatches.

In similar fashion, DNA probes can be used to detect mismatches, throughenzymatic or chemical cleavage. See, e.g., Crotton et al., Proc. Natl.Acad. Sci. USA, Vol. 85, 4397, 1988; and Shenk et al., Proc. Natl. Acad.Sci. USA, Vol. 72, p. 989; 1975. Alternatively, mismatches can bedetected by shifts in the electrophoretic mobility of mismatchedduplexes relative to matched duplexes. See, e.g., Cariello, HumanGenetics, Vol. 42, p. 726, 1988. With either riboprobes or DNA probes,the cellular mRNA or DNA which might contain a mutation can be amplifiedusing PCR (see below) before hybridization. Changes in DNA of the APCgene can also be detected using Southern hybridization, especially ifthe changes are gross rearrangements, such as deletions and insertions.

DNA sequences of the APC gene which have been amplified by use ofpolymerase chain reaction may also be screened using allele-specificprobes. These probes are nucleic acid oligomers, each of which containsa region of the APC gene sequence harboring a known mutation. Forexample, one oligomer may be about 30 nucleotides in length,corresponding to a portion of the A PC gene sequence. By use of abattery of such allele-specific probes, PCR amplification products canbe screened to identify the presence of a previously identified mutationin the APC gene. Hybridization of allele-specific probes with amplifiedAPC sequences can be performed, for example, on a nylon filter.Hybridization to a particular probe under stringent hybridizationconditions indicates the presence of the same mutation in the tumortissue as in the allele-specific probe.

Alteration of APC mRNA expression can be detected by any technique knownin the art. These include Northern blot analysis, PCR amplification andRNase protection. Diminished mRNA expression indicates an alteration ofthe wild-type APC gene. Alteration of wild-type APC genes can also bedetected by screening for alteration of wild-type APC protein. Forexample, monoclonal antibodies immunoreactive with APC can be used toscreen a tissue. Lack of cognate antigen would indicate an APC mutation.Antibodies specific for products of mutant alleles could also be used todetect mutant APC gene product. Such immunological assays can be done inany convenient format known in the art. These include Western blots,immunohistochemical assays and ELISA assays. Any means for detecting analtered APC protein can be used to detect alteration of wild-type APCgenes. Functional assays can be used, such as protein bindingdeterminations. For example, it is believed that APC proteinoligomerizes to itself and/or MCC protein or binds to a G protein. Thus,an assay for the ability to bind to wild type APC or MCC protein or thatG protein can be employed. In addition, assays can be used which detectAPC biochemical function. It is believed that APC is involved inphospholipid metabolism. Thus, assaying the enzymatic products of theinvolved phospholipid metabolic pathway can be used to determine APCactivity. Finding a mutant APC gene product indicates alteration of awild-type APC gene.

Mutant APC gene or gene products can also be detected in other humanbody samples, such as, serum, stool, urine and sputum. The sametechniques discussed above for detection of mutant APC genes or geneproducts in tissues can be applied to other body samples. Cancer cellsare sloughed off from tumors and appear in such body samples. Inaddition, the APC gene product itself may be secreted into theextracellular space and found in these body samples even in the absenceof cancer cells. By screening such body samples, a simple earlydiagnosis can be achieved for many types of cancers. In addition, theprogress of chemotherapy or radiotherapy can be monitored more easily bytesting such body samples for mutant APC genes or gene products.

The methods of diagnosis of the present invention are applicable to anytumor in which APC has a role in tumorigenesis. Deletions of chromosomearm 5q have been observed in tumors of lung, breast, colon, rectum,bladder, liver, sarcomas, stomach and prostate, as well as in leukemiasand lymphomas. Thus these are likely to be tumors in which APC has arole. The diagnostic method of the present invention is useful forclinicians so that they can decide upon an appropriate course oftreatment. For example, a tumor displaying alteration of both APCalleles might suggest a more aggressive therapeutic regimen than a tumordisplaying alteration of only one APC allele.

The primer pairs of the present invention are useful for determinationof the nucleotide sequence of a particular APC allele using thepolymerase chain reaction. The pairs of single stranded DNA primers canbe annealed to sequences within or surrounding the APC gene orchromosome 5q in order to prime amplifying DNA synthesis of the APC geneitself. A complete set of these primers allows synthesis of all of thenucleotides of the APC gene coding sequences, i.e., the exons. The setof primers preferably allows synthesis of both intron and exonsequences. Allele specific primers can also be used. Such primers annealonly to particular APC mutant alleles, and thus will only amplify aproduct in the presence of the mutant allele as a template.

In order to facilitate subsequent cloning of amplified sequences,primers may have restriction enzyme site sequences appended to their 5′ends. Thus, all nucleotides of the primers are derived from APCsequences or sequences adjacent to APC except the few nucleotidesnecessary to form a restriction enzyme site. Such enzymes and sites arewell known in the art. The primers themselves can be synthesized usingtechniques which are well known in the art. Generally, the primers canbe made using oligonucleotide synthesizing machines which arecommercially available. Given the sequence of the APC open reading frameshown in FIG. 3 FIGS. 3A-3Z (SEQ ID NO:1), design of particular primersis well within the skill of the art.

The nucleic acid probes provided by the present invention are useful fora number of purposes. They can be used in Southern hybridization togenomic DNA and in the RNase protection method for detecting pointmutation already discussed above. The probes can be used to detect PCRamplification products. They may also be used to detect mismatches withthe APC gene or mRNA using other techniques. Mismatches can be detectedusing either enzymes (e.g., S1 nuclease), chemicals (e.g., hydroxylamineor osmium tetraoxide and piperidine), or changes in electrophoreticmobility of mismatched hybrids as compared to totally matched hybrids.These techniques are known in the art. See, Cotton, supra, Shenk, supra,Myers, supra, Winter, supra, and Novack et al., Proc. Natl. Acad. Sci.USA, Vol. 83, p. 586, 1986. Generally, the probes are complementary toAPC gene coding sequences, although probes to certain introns are alsocontemplated. An entire battery of nucleic acid probes is used tocompose a kit for detecting alteration of wild-type APC genes. The kitallows for hybridization to the entire APC gene. The probes may overlapwith each other or be contiguous.

If a riboprobe is used to detect mismatches with mRNA, it iscomplementary to the mRNA of the human wild-type APC gene. The riboprobethus is an anti-sense probe in that it does not code for the APC proteinbecause it is of the opposite polarity to the sense strand. Theriboprobe generally will be labeled with a radioactive, colorimetric, orfluorometric material, which can be accomplished by any means known inthe art. If the riboprobe is used to detect mismatches with DNA it canbe of either polarity, sense or anti-sense. Similarity, DNA probes alsomay be used to detect mismatches.

Nucleic acid probes may also be complementary to mutant alleles of theAPC gene. These are useful to detect similar mutations in other patientson the basis of hybridization rather than mismatches. These arediscussed above and referred to as allele-specific probes. As mentionedabove, the A PC probes can also be used in Southern hybridizations togenomic DNA to detect gross chromosomal changes such as deletions andinsertions. The probes can also be used to select cDNA clones of APCgenes from tumor and normal tissues. In addition, the probes can be usedto detect APC mRNA in tissues to determine if expression is diminishedas a result of alteration of wild-type APC genes.

According to the present invention a method is also provided ofsupplying wild-type APC function to a cell which carries mutant APCalleles. Supplying such function should suppress neoplastic growth ofthe recipient cells. The wile-type APC gene or a part of the gene may beintroduced into the cell in a vector such that the gene remainsextrachromosomal. In such a situation the gene will be expressed by thecell from the extrachromosomal location. If a gene portion is introducedand expressed in a cell carrying a mutant APC allele, the gene portionshould encode a part of the APC protein which is required fornon-neoplastic growth of the cell. More preferred is the situation wherethe wild-type APC gene or a part of it is introduced into the mutantcell in such a way that it recombines with the endogenous mutant APCgene present in the cell. Such recombination requires a doublerecombination event which results in the correction of the APC genemutation. Vectors for introduction of genes beth both for recombinationand for extrachromosomal maintenance are known in the art and anysuitable vector may be used. Methods for introducing DNA into cells suchas electroporation, calcium phosphate co-precipitation and viraltransduction are known in the art and the choice of method is within thecompetence of the routineer. Cells transformed with the wild-type A PCAPC gene can be used as model systems to study cancer remission and drugtreatments which promote such remission.

Similarly, cells and animals which carry a mutant APC allele can be usedas model systems to study and test for substances which have potentialas therapeutic agents. The cells are typically cultured epithelialcells. These may be isolated from individuals with APC mutations, eithersomatic or germline. Alternatively, the cell line can be engineered tocarry the mutation in the APC allele. After a test substance is appliedto the cells, the neoplastically transformed pheno-type phenotype of thecell will be determined. Any trait of neoplastically transformed cellscan be assessed, including anchorage-independent growth, tumorigenicityin nude mice, invasiveness of cells, and growth factor dependence.Assays for each of these traits are known in the art.

Animals for testing therapeutic agents can be selected after mutageneisof whole animals or after treatment of germline cells or zygotes. Suchtreatments include insertion of mutant A PC alleles, usually from asecond animal species, as well as insertion of disrupted homologousgenes. Alternatively, the endogenous APC gene(s) of the animals may bedisrupted by insertion or deletion mutation. After test substances havebeen administered to the animals, the growth of tumors must be assessed.If the test substance prevents or suppresses the growth of tumors, thenthe test substance is a candidate therapeutic agent for the treatment ofFAP and/or sporadic cancers.

Polypeptides which have APC activity can be supplied to cells whichcarry mutant or missing APC alleles. The sequence of the APC protein isdisclosed in FIG. 3 FIGS. 3A-3Z (SEQ ID NO:7). These two sequencesdiffer slightly and appear to be indicate the existence of two differentforms of the APC protein. Protein can be produced by expression of thecDNA sequence in bacteria, for example, using known expression vectors.Alternatively, APC can be extracted from APC-producing mammalian cellssuch as brain cells. In addition, the techniques of synthetic chemistrycan be employed to synthesize APC protein. Any of such techniques canprovide the preparation of the present invention which comprises the APCprotein. The preparation is substantially free of other human proteins.This is most readily accomplished by synthesis in a microorganism or invitro.

Active APC molecules can be introduced into cells by microinjection orby use of liposomes, for example. Alternatively, some such activemolecules may be taken up by cells, actively or by diffusion.Extracellular application of APC gene product may be sufficient toaffect tumor growth. Supply of molecules with APC activity should leadto a partial reversal of the neoplastic state. Other molecules with APCactivity may also be used to effect such a reversal, for examplepeptides, drugs, or organic compounds.

The present invention also provides a preparation of antibodiesimmunoreactive with a human APC protein. The antibodies may bepolyclonal or monoclonal and may be raised against native APC protein,APC fusion proteins, or mutant APC proteins. The antibodies should beimmunoreactive with APC epitopes, preferably epitopes not present onother human proteins. In a preferred embodiment of the invention theantibodies will immunoprecipitate APC proteins from solution as well asreact with APC protein on Western or immunoblots of polyacrylamide gels.In another preferred embodiment, the antibodies will detect APC proteinsin paraffin or frozen tissue sections, using immunocytochemicaltechniques. Techniques for raising and purifying antibodies are wellknown in the art and any such techniques may be chosen to achieve thepreparation of the invention.

Predisposition to cancers as in FAP and GS can be ascertained by testingany tissue of a human for mutations of the APC gene. For example, aperson who has inherited a germline APC mutation would be prone todevelop cancers. This can be determined by testing DNA from any tissueof the persons's body. Most simply, blood can be drawn and DNA extractedfrom the cells of the blood. In addition, prenatal diagnosis can beaccomplished by testing fetal cells, placental cells, or amniotic fluidfor mutation of the APC gene. Alteration of a wild-type APC allele,whether for example, by point mutation or by deletion, can be detectedby any of the means discussed above.

Molecules of cDNA according to the present invention are intron-free,APC gene ceding molecules. They can be made by reverse transcriptaseusing the APC mRNA as a template. These molecules can be propagated invectors and cell lines as is known in the art. Such molecules have thesequence shown in SEQ ID NO:3. The cDNA can also be made using thetechniques of synthetic chemistry given the sequence disclosed herein.

A short region of homology has been identified between APC and the humanm3 muscarinic acetylcholine receptor (mAChR). This homology was largelyconfined to 29 residues in which 6 out of 7 amino acids (EL(GorA)GLQA)were identical (See FIG. 4 FIG. 4B (SEQ ID NO: 9)). Initially, it wasnot known whether this homology was significant, because many otherproteins had higher levels of global homology (though few had six out ofseven contiguous amino acids in common). However, a study on thesequence elements controlling G protein activation by mAChR subtypes(Lechleiter et al., EMBO J., p. 4381 (1990)) has shown that a 21 aminoacid region from the m3 mAChR completely mediated G protein specificitywhen substituted for the 21 amino acids of m2 mA ChR at the analogousprotein position. These 21 residues overlap the 19 amino acid homologybetween APC and m3 mA ChR.

This connection between APC and the G protein activating region of themAChR is intriguing in light of previous investigations relating Gproteins to cancer. For example, the RAS oncogenes, which are oftenmutated in colorectal cancers (Vogelstein, et al., N. Engl. J. Med.,Vol. 319, p. 525 (1988); Bos et al., Nature Vol. 327, p. 293 (1987)),are members of the (1 protein family (Bourne, et al, Nature, Vol. 348,p. 125 (1990)) as is an in vitro transformation suppressor (Noda et al.,Proc. Natl. Acad. Sci. USA, Vol. 86, p. 162 (1989)) and genes mutated inhormone producing tumors (Candis et al., Nature, vol. 340, p. 692(1989); Lyons et al., Science, Vol. 249, p. 655 (1990)). Additionally,the gene responsible for neurofibromatosis (presumably a tumorsuppressor gene) has been shown to activate the GTPase activity of RAS(Xu et al., Cell, Vol. 63, p. 835 (1990); Martin et al., Cell, Vol. 63,p. 843 (1990); Ballester et al., Cell, Vol. 63, p. 851 (1990)). Anotherinteresting link between G proteins and colon cancer involves the drugsulindac. This agent has been shown to inhibit the growth of benigncolon tumors in patients with FAP, presumably by virtue of its activityas a cyclooxygenase inhibitor (Waddell et al., J. Surg. Oncolong 24(1),83 (1983); Wadell et al., Am. J. Surg., 157(1), 175 (1989); Charneau etal., Gastroenterologie Clinique at Biologique 14(2), 153 (1990)).Cyclooxygenase is required to convert arachidonic acid to prostaglandisand other biologically active molecules. G proteins are known toregulate phospholipase A2 activity, which generates arachidonic acidfrom phospholipids (Role et al., Proc. Natl. Acad. Sci. USA, Vol. 84, p.3623 (1987); Kurachi et al., Nature, Vol. 337, 12 555 (1989)). Thereforewe propose that wild-type APC protein functions by interacting with a Gprotein and is involved in phospholipid metabolism.

The following are provided for exemplification purposes only and are notintended to limit the scope of the invention which has been described inbroad terms above.

EXAMPLE 1

This example demonstrates the isolation of a 5.5 Mb region of human DNAlinked to the FAP locus. Six genes are identified in this region, all ofwhich are expressed in normal colon cells and in colorectal, lung, adand bladder tumors.

The cosmid markers YN5.64 and YN5.48 have previously been shown todelimit an 8 cM region containing the locus for FAP (Nakamura et al.,Am. J. Hum. Genet. Vol. 43, p. 638 (1988)). Further linkage andpulse-field gel electrophoresis (PFGE) analysis with additional markershas shown that the FAP locus is contained within a 4 cM region borderedby cosmids EF5.44 and L5.99. In order to isolate clones representing asignificant portion of this locus, a yeast artificial chromosome (YAC)library was screened with various 5q21 markers. Twenty-one YAC clones,distributed within six contigs and including 5.5 Mb from the regionbetween YN5.64 and YN5.48, were obtained (FIG. 1A).

Three contigs encompassing approximately 4 Mb were contained within thecentral portion of this region. The YAC's YACs constituting thesecontigs, together with the markers used for their isolation andorientations, are shown in FIG. 1. These YAC contigs were obtained inthe following way. To initiate each contig, the sequence of a genomicmarker cloned from chromosome 5q21 was determined and used to designprimers for PCR. PCR was then carried out on pools of YAC clonesdistributed in microtiter trays as previously described (Anand et al.,Nucleic Acids Research, Vol. 18, p. 1951 (1980)). Individual YAC clonesfrom the positive pools were identified by further PCR or hybridizationbased assays, and the YAC sizes were determined by PFGE.

To extend the areas covered by the original YAC clones, “chromosomalwalking” was performed. For this purpose, YAC termini were isolated by aPCR based method and sequenced (Riley et al., Nucleic Acids Research,Vol. 18, p. 2887 (1990)). PCR primers based on these sequences were thenused to rescreen the YAC library. For example, the sequence from anintron of the FER gene (Hao et al., Mol. Cell. Biol., Vol. 9, p. 1587(1989)) was used to design PCR primers for isolation of the 28EC1 and5EH8 YACs. The termini of the 28EC1 YAC were sequenced to derive markersRHE28 and LHE28, respectively. The sequences of these two markers werethen used to isolate YAC clones 15CH12 (from RHE28) and 40CF1 and 29EF1(from LHE28). These five YAC's YACs formed a contig encompassing 1200 kb(contig 1, FIG. 1B).

Similarly, contig 2 was initiated using cosmid N5.66 sequences, andcontig 3 was initiated using sequences both from the MCC gene and fromcosmid EF5.44. A walk in the telomeric direction from YAC 14FH1 and awalk in the opposite direction from YAC39GG3 allowed connection of theinitial contig 3 clones through YAG 37HG4 (FIG. 1B). YAC37HG4 wasdeposited at the National Collection of Industrial and Marine Bacteria(NCIMB), P.O. Box 31, 23 St. Machar Drive, Aberdeen AB2 1RY, Scotland,under Accession No. 40353 on Dec. 17, 1990.

Multipoint linkage analysis with the various markers used to define thecontigs, combined with PPGE analysis, showed that contigs 1 and 2 werecentromeric to contig 3. These contigs were used as tools to orientand/or identify genes which might be responsible for FAP. Six genes werefound to lie within this cluster of YAC's YACs, as follows:

Contig #1: FER—The FER gene was discovered through its homology to theviral oncogene. ABL (Hao et al., supra). It has an intrinsic tyrosinekinase activity, and in situ hybridization with an FER probe showed thatthe gene was located at 5q11-23 (Morris et al., Cytogenet. Cell. Genet.,Vol. 53, p. 4, (1990)). Because of the potential role of thisoncogene-related gene in neoplasia, we decided to evaluate it furtherwith regards to the FAP locus. A human genomic clone from FER wasisolated (MF 2.3) and used to define a restriction fragment lengthpolymorphism (RFLP), and the RFLP in turn used to map FER by linkageanalysis using a panel of three generation families. This showed thatFER was very tightly linked to previously defined polymorphic markersfor the FAP locus. The genetic mapping of FER was complemented byphysical mapping using the YAC clones derived from FER sequences (FIG.1B). Analysis of YAC contig 1 showed that FER was within 600 kb ofcosmid marker M5.28, which maps to within 1.5 Mb of cosmid L5.99 by PFGEof human genomic DNA. Thus, the YAC mapping results were consistent withthe FER linkage data and PFGE analyses.

Contig 2: TB1—TB1 was identified through a cross-hybridization approach.Exons of genes are often evolutionarily conserved while introns andintergenie intergenic regions are much less conserved. Thus, it a humanprobe cross-hybridizes strongly to the DNA from the non-primate species,there is a reasonable chance that it contains exon sequences. Subclonesof the cosmids shown in FIG. 1 were used to screen Southern blotscontaining rodent DNA samples. A subclones of cosmid N5.66 (p 5.66-4)was shown to strongly hybridize to rodent DNA, and this clone was usedto screen cDNA libraries derived from normal adult colon and fetalliver. The ends of the initial cDNA clones obtained in this screen werethen used to extend the cDNA sequence. Eventually, 11 cDNA clones wereisolated, covering 2314 bp. The gene detected by these clones was namedTB1. Sequence analysis of the overlapping clones revealed an openreading frame (ORF) that extended for 1302 bp starting from the most 5′sequence data obtained (FIG. 2A). If this entire open reading frame weretranslated, it would encode 434 amino acids (SEQ ID NO:5). The productof this gene was not globally homologous to any other sequence in thecurrent database but showed two significant local similarities to afamily of ADP,ATP carrier/translocator proteins and mitochondrial brownfat uncoupling proteins which are widely distributed from yeast tomammals. These conserved regions of TB1 (underlined in FIG. 2A) maydefine a predictve motif for this sequence family. In addition, TB1appeared to contain a signal peptide (or mitochondrial targetingsequence) as well as at least 7 transmembrane domains.

Contig 3: MCC, TB2, SRP and APC—The MCC gene was also discovered througha cross-hydridization approach, as described previously (Kinzler et al.,Science Vol. 251, p. 1366 (1991)). The MCC gene was considered acandidate for causing FAP by virtue of its tight genetic linkage to FAPsusceptibility and its somatic mutation in sporadic colorectalcarcinomas. However, mapping experiments suggested that the cedingregion of MCC was approximately 50 kb to proximal to the centromeric endof a 200 kb deletion found in an FAP patient. MCC cDNA probes detected a10 mb mRNA transcript on Northern blot analysis of which 4151 bp,including the entire open reading frame, have been cloned. Although the3′ non-translated portion or an alternatively spliced form of MCC mighthave extended into this deletion, it was possible that the deletion didnot affect the MCC gene product. We therefore used MCC sequences toinitiate a YAC contig, and subsequently used the YAC clones to identifygenes 50 to 250 kb distal to MCC that might be contained within thedeletion.

In a first approach, the insert from YAC24ED6 (FIG. 1B) wasradiolabelled and hybridized to a cDNA library from normal colon. One ofthe cDNA clones (YS39) identified in this manner detected a 3.1 kb mRNAtranscript when used as a probe for Northern blot hybridization.Sequence analysis of the YS39 clone revealed that it encompassed 2283nucleotides and contained an ORF that extended for 555 bp from the most5′ sequence data obtained. If all of this ORF were translated, it wouldencode 185 amino acids (SEQ ID NO:6) (FIG. 2B). The gene detected byYS39 was named TB2. Searches of nucleotide and protein database revealedthat the TB2 gene was not identical to any previously reported sequencesnor were there any striking similarities.

Another clone (YS11) identified through the YAC 24ED6 screen appeared tocontain portions of two distinct genes. Sequences from one end of YS11were identical to at least 180 bp of the signal recognition particleprotein SRP19 (Lingelbach et al. Nucleic Acids Research, Vol. 16, p.9431 (1988). A second ORF, from the opposite end of clone YS11, provedto be identical to 78 bp of a novel gene which was independentlyidentified through a second YAC-based approach. For the latter, DNA fromyeast cells containing YAC 14FH1 (FIG. 1B) was digested with EcoRI andsub-cloned into a plasmid vector. Plasmids that contained human DNAfragments were selected by colony hybridization using total human DNA asa probe. These clones were then used to search for cross-hybridizingsequences as described above for TB1, and the cross-hybridizing cloneswere subsequently used to screen cDNA libraries. One of the cDNA clonesdiscovered in this way (FH38) contained a long ORF (2496 bp), 78 bp ofwhich were identical to the above-noted sequences in YS11. The ends ofthe FH38 cDNA clone were then used to initiate cDNA walking to extendthe sequence. Eventually, 85 cDNA clones were isolated from normalcolon, brain and liver cDNA libraries and found to encompass 8973nucleotides of contiguous transcript. The gene corresponding to theirtranscript was named APC. When used as probes for Northern blotanalysis, APC cDNA clones hybridized to a single transcript ofapproximately 9.5 kb, suggesting that the great majority of the geneproduct was represented in the cDNA clones obtained. Sequences from the5′ end of the APC gene were found in YAC 37HG4 but not in YAC 14H1.However, the 3′ end of the APC gene was found in 14FH1 as well as 37HG4.Analogously, the 5′ end of the MCC ceding region was found in YAC clones19AA9 and 266C3 but not 24ED6 or 14FH1, while the 3′ end displayed theopposite pattern. Thus, MCC and APC transcription units pointed inopposite directions, with the direction of transcription going fromcentromeric to telometric in the case of MCC, and telomeric tocentromeric in the case of APC. PFGE analysis of YAC DNA digested withvarious restriction endonucleases showed that TB2 and SRP were betweenMCC and APC, and that the 3′ ends of the ceding regions of MCC and APCwere separated by approximately 150 kb (FIG. 1B).

Sequence analysis of the APC cDNA clones revealed an open reading frameof 8,535 nucleotides. The 5′ end of the ORF contained a methionine codon(codon 1) that was preceded by an in-frame stop codon 9 bp upstream, andthe 3′ end was followed by several in-frame stop codons. The proteinproduced by initiation at codon 1 would contain 2,842 2843 amino acids(SEQ ID NO:7) (FIG. 3) FIG. 3A-3Z. The results of database searchingwith the APC gene product were quite complex due to the presence oflarge segments with locally biased amino acid compositions. In spite ofthis, APC could be roughly divided into two domains. The N-terminal 25%of the protein had a high content of leucine residue (12%) and showedlocal sequence similarities to myosins, various intermediate filamentproteins (e.g., desmin, vimentin, neuroflilaments) and Drospophilaarmadillo/human plakeoglobin. The latter protein is a component ofadhesive junctions (desmosomes) joining epithelial cells (Franke et al.,Proc. Natl. Acad. Sci. U.S.A., Vol. 86, p. 4027 (1989); Perfer et al.,Cell, Vol. 63, p. 1167 (1990)) The C-terminal 75% of APC (residues731-2832) is 17% serine by composition with serine residues more or lessuniformly distributed. This large domain also contains localconcentrations of charged (mostly acidic) and proline residues. Therewas no indication of potential signal peptide, transmembrane regions, ornuclear targeting signals in APC, suggesting a cytoplasmic localization.

To detect short similarities to APC, a database search was performedusing the PAM-40 matrix (Altschul. J. Mol. Biol., Vol. 219, p. 555(1991). Potentially interesting matches to several proteins were found.The most suggestive of these involved the ral2 gene product of yeast,which is implicated in the regulation of ras activity (Fukul et al.,Mol. Cell. Biol., Vol. 9, p. 5617 (1989)). Little is known about howral2 might interact with rts but it is interesting to note thepositively-charged character of this region in the context of thenegatively-charged GAP interaction region of ras. A specificelectrostatic interaction between ras and GAP-related proteins has beenproposed.

Because of the proximity of the MCC and APC genes, and the fact thatboth am are implicated in colorectal tumorigenesis, we searched forsimilarities between the two predicted proteins. Bourne has previouslynoted that MCC has the potential to form alpha helical coiled coils(Nature, Vol. 351, p. 188 (1991). Lupas and colleagues have recentlydeveloped a program for predicting coiled coil potential from primarysequence data (Science, Vol. 252, p. 1162 (1991) and we have used theirprogram to analyze both MCC and APC. Analysis of MCC indicated adiscontinuous pattern of coiled-coil domains separated by putative“hinge” or “sparcer” regions similar to those seen in laminin and otherintermediate filament proteins. Analysis of the APC sequence revealedtwo regions in the N-terminal domain which had strong coiledcoil-forming potential, and these regions corresponded to those thatshowed local similarities with myosin and IF proteins on databasesearching. In addition, one other putative coiled coil region wasidentified in the central region of the APC. The potential for both APCand MCC to form coiled coils is interesting in that such structuresoften mediate homo- and hetero-oligomerization heterooligomerization.

Finally, it had previously been noted that MCC shared a short similaritywith the region of the m3 muscarinic acetylcholine receptor (mAChR)known to regulate specificity of G-protein coupling. The APC gene alsocontained a local similarity to the region of the m3 mAChR (SEQ ID NO:9)that overlapped with the MCC similarity (SEQ ID NO:10) (FIG. 4B).Although the similarities to ral2 (SEQ ID NO:8) (FIG. 4A) and m3 mACHR(SEQ ID NO:9) (FIG. 4B) were not statistically significant, they wereintriguing in light of previous observations resulting G-proteins toneoplasia.

Each of the six genes described above was expressed in normal colonmucosa, as indicated by their representation in colon cDNA libraries. Tostudy expression of the genes in neoplastic colorectal epithelium, weemployed reverse transcription-polymerase chain reaction (PCR) assays.Primers based on the sequences of FER, TB1, TB2, MCC, and APC were eachused to design primers for PCR performed with cDNA templates. Each ofthese genes was found to be expressed in normal colon, in each of tencell lines derived from colorectal cancers, and in tumor cell linesderived from lung and bladder tumors. The ten colorectal cancer celllines included eight from patients with sporadic CRC and two frompatients with FAP.

EXAMPLE 2

This example demonstrates a genetic analysis of the role of the FER genein FAP and sporadic colorectal cancers.

We considered FER as a candidate because of its proximity to the FAPlocus as judged by physical and genetic criteria (see Example 1), andits homology to known tyrosine kinases with oncogenic potential. Primerswere designed to PCR-amplify the complete coding sequence of FER fromthe RNA of two colorectal cancer cell lines derived from FAP patients.cDNA was generated from RNA and used as a template for PCR. The primersused were 5′-AGAAGGATCCCTTGTGCAGTGTGGA-3′ (SEQ ID NO:95) and5′-GACAGGATCCTGAAGCTGAGTTTG-3′ (SEQ ID NO:96). The underlinednucleotides were altered from the true FER sequence to create BamHIsites. The cell lines used were JW and Difi, both derived fromcolorectal cancers of FAP patients. (C. Parkaskeva, B. G. Buckle, D.Sheer, C. B. Wigley, Int. J. Cancer 34, 49 (1984); M. E. Gross et al.,Cancer Res. 51, 1452 (1991). The resultant 2554 basepair fragments werecloned and sequences in their entirety. The PCR products were cloned inthe BamHI site of Bluescript SK (Stratagene) and pools of at least 50clones were sequenced en masse using T7 polymerase, as described inNigro et al., Nature 342, 705 (1989).

Only a single conservative amino acid change (GTG→CTG, creating a val toleu substitution at codon 439) was observed. The region surrounding thiscodon was then amplified from the DNA of individuals without FAP andthis substitution was found to be a common polymorphism, notspecifically associated with FAP. Based on these results, we consideredit unlikely (though still possible) the FER gene was responsible forFAP. To amplify the regions surrounding codon 439, the following primerswere used: 5′-TCAGAAAGTGCTGAAGAG-3′ (SEQ ID NO:97) and5′-GGAATAATTAGGTCTCCAA-3′ (SEQ ID NO:98). PCR products were digestedwith PstI, which yields a 50 bp fragment if codon 439 is leucine, but 26and 24 bp fragments if it is valine. The primers used for sequencingwere chosen from the FER cDNA sequence in Hao et al., supra.

EXAMPLE 3

This example demonstrates the genetic analysis of MCC, TB2, SRP and ACPin FAP and sporadic colorectal tumors. Each of these genes is linked andencompassed by contig 3 (see FIG. 1).

Several lines of evidence suggested that this contig was of particularinterest. First, at least three of the four genes in this contig werewithin the deleted region identified in two FAP patients. (See Example 5infra.) Second, allelic deletions of chromosome 5q21 in sporadic cancersappeared to be centered in this region. (Ashton-Rickardt et al.,Oncogene, in press; and Miki et al., Japn. J. Cancer Res., in press.)Some tumors exhibited loss of proximal RFLP markers (up to andpotentially including the 5′ end of MCC), but no loss of markers distalto MCC. Other tumors exhibited loss of markers distal to and perhapsincluding the 3′ end of MCC, but no loss of sequences proximal to MCC.This suggested either that different ends of MCC were affected by lossin all such cases, or alternatively, that two genes (one proximal to andperhaps including MCC, the other distal to MCC) were separate targets ofdeletion. Third, clones from each of the six FAP region genes were usedas probes on Southern blots containing tumor DNA from patients withSporadic CRC. Only two examples of somatic changes were observed in over200 tumors studied; a rearrangement/deletion whose centromeric end waslocated within the MCC gene (Kinzler et al., supra) and an 800 bpinsertion within the APC gene between nucleotides 4424 and 5584. Fourth,point mutations of MCC were observed in two tumors (Kinzler et al.)supra strongly suggesting that MCC was a target of mutation in at leastsome sporadic colorectal cancers.

Based on these results, we attempted to search for subtle alterations ofcontig 3 genes in patients with FAP. We chose to examine MCC and APC,rather than TB2 or SRP, because of the somatic mutations in MCC and APCnoted above. To facilitate the identification of subtle alteration, thegenomic sequences of MCC and APC exons were determined (see Table I, SEQID NO:24-38).

TABLE I APC EXONS EXON NUCLEOTIDES¹ EXON BOUNDARY SEQUENCE²  822 to 930catgatgttatctgtatttacctatagtctaaattataccatctataatgtgcttaatttttag/GGTTAA. . . (SEQ ID NO:24) . . .ACCAAG/gtaacagaagattacaaaccctggtcactaatgccatgactactttgctaag (SEQ IDNO:25)  931 to 1309 ggatattaagtcgtaattttgtttctaaactcatttggcccacag/GTGGAA. . . (SEQ ID NO:26) . . . ATCCAA/gtatgttctctatagtgtacattcgtagtgcatg(SEQ ID NO:27) 1310 to 1405catcattgctcttcaaataacaaagcattatggtttatgttgatttatttttcag/TGCCAG . . .(SEQ ID NO:28) . . . AACTAG/gtaagacaaaaatgttttttaatgacatagacaattactggtg(SEQ ID NO:29) 1406 to 1545tagatgattgtctttttcctcttgccctttttaaattag/GGGGAC . . . (SEQ ID NO:30) . .. AACAAG/gtatgtttttataacatgtatttcttaaggatagctcaggtatga (SEQ ID NO:31)1546 to 1623gcttggcttcaagttgtctttttaatgatcctctattctgtatttaatttacag/GCTACG . . . (SEQID NO:32) . . .CAGCAG/gtactatttagaatttcacctgtttttcttttttctctttttctttgaggcagggtctcactctg(SEQ ID NO:33) 1624 to 1740gcaactagtatgattttatgtataaattaatctaaaattgatttgacag/GTTATT . . . (SEQ IDNO:34) . . . AAAAAG/gtacctttgaaaacatttagtactataatatgaatttcatgt (SEQ IDNO:35) 1741 to 1955 caactctaattagatgacccatattcagaaacttactag/GAATCA . . .(SEQ ID NO:36) . . .CCACAG/gtatatatagagttttatattacttttaaagtacagaattcatactctcaaaaa (SEQ IDNO:37) 1956 to 8973 tcttgattttttatttcag/GCAAAT . . . (SEQ ID NO:38) . .. GGTATTTATGCAAAAAAAAATGTTTTTGT (SEQ ID NO:1) ¹Relative to predictedtranslation initiation site ²Small case letters represent introns, largecase letters represent exons The entire 5′ end of the cloned APC cDNA(at 1956-8973) appeared to be encoded in this exon, as indicated byrestriction endonuclease mapping and sequencing of the cloned genomicDNA. The ORF ended at nt 8535. The extreme 3′ end of the APC transcripthas not yet been identified.These sequences were used to design primers for PCR analysis ofconstitutional DNA from FAP patients.

We first amplified eight exons and surrounding introns of the MCC genein affected individuals from 90 different FAP kindreds. The PCR productswere analyzed by a ribonuclease (RNase) protein assay. In brief, the PCRproducts were hybridized to in vitro transcribed RNA probes representingthe normal genomic sequences. The hybrids were digested with RNase A,which can cleave at single base pair mismatches within DNA-RNA hybrids,and the cleavage products were visualized following denaturing gelelectrophoresis. Two separate RNase protection analyses were performedfor each exon, one with the sense and one with the antisense strand.Under these conditions, approximately 40% of all mismatches aredetectable. Although some amino acid variants of MCC were observed inFAP patients, all such variants were found in a small percentage ofnormal individuals. These variants were thus unlikely to be responsiblefor the inheritance of FAP.

We next examined three exons of the A PC APC gene. The three exonsexamined included those containing nt 822-930, 931-1309, and the first300 nt of the most distal exon (nt 1956-2256). PCR and RNase protectionanalysis were performed as described in Kinzler et al. supra, using theprimers underlined in Table I (SEQ ID NO:24-38). The primers for nt1956-2256 were 5′-GCAAATCCTAAGAGAGAACAA-3′ (SEQ ID NO:99) and5′-GATGGCAAGCTTGAGCCAG-3′ (SEQ ID NO:100).

In 90 kindreds, the RNase protection method was used to screen formutations and in an additional 13 kindreds, the PCR products were clonedand sequenced to search for mutations not detectable by RNaseprotection. PCR products were cloned into a Bluescript vector modifiedas described in T. A. Holton and M. W. Graham, Nucleic Acid Res. 19,1156 (1991). A minimum of 100 clones were pooled and sequenced. Fivevariants were detected among the 103 kindreds analyzed. Cloning andsubsequent DNA sequencing of the PCR product of patient P21 indicated aC to T transition in codon 413 that resulted in a change from arginineto cysteine. This amino acid variant was not observed in any of 200 DNAsamples from individuals without FAP. Cloning and sequencing of the PCRproduct from patients P24 and P34, who demonstrated the same abnormalRNase protection pattern indicated that both had a C to T transition atcodon 801 that resulted in a change from arginine (CGA) to a stop codon(TGA). This change was not present in 200 individuals without FAP. Asthis point mutation resulted in the predicted loss of the recognitionsite for the enzyme Taq I, appropriate PCR products could be digestedwith Taq I to detect the mutation. This allowed us to determine that thestop codon co-segregated cosegregated with disease phenotype in membersof the family of P24. The inheritance of this change in affected membersof the pedigree provides additional evidence for the importance of themutation.

Cloning and sequencing of the PCR product from FAP patient P93 indicateda C to G transversion of codon 279, also resulting in a stop codon(change from TCA to TGA). This mutation was not present in 200individuals without FAP. Finally, one additional mutation resulting in aserine (TCA) to stop codon (TGA) at codon 712 was detected in a singlepatient with FAP (patient P60).

The five germline mutations identified are summarized in Table IIA, aswell as four others discussed in Example 9.

TABLE IIA Germline mutations of the APC gene in FAP and GS PatientsEXTRA- COLO- NIC NUCLEO- AMINO PATIENT TIDE ACID DISEASE CODON CHANGECHANGE AGE 93 279 TCA->TGA Ser->Stop 39 Mandi- bular Osteoma 24 301CGA->TGA Arg->Stop 46 None 34 301 CGA->TGA Arg->Stop 27 Des- moid Tumor21 413 CGC->TGC Arg->Cys 24 Mandi- bular Osteoma 60 712 TCA->TGASer->Stop 37 Mandi- bular Osteoma 3746 243 CAGAG->CAG splice- junction3460 301 CGA->TGA Arg->Stop 3827 456 CTTTCA->CTTCA frameshift 3712 500T->G Tyr->Stop *The mutated nucleotides are underlined.In addition to these germline mutations, we identified several somaticmutations of MCC and APC in sporadic CRC's S CRC'CRCs. Seventeen MCCexons were examined in 90 sporadic colorectal cancers by RNAseprotection analysis. In each case where an abnormal RNAse protectionpattern was observed, the corresponding PCR products were cloned andsequenced. This led to the identification of six point mutations (twodescribed previously) (Kinzler et al., supra), each of which was notfound in the germline of these patients (Table IIB).

TABLE IIB Somatic Mutations in Sporadic CRC Patients AMINO NUCLEOTIDEACID PATIENT CODON¹ CHANGE CHANGE T35 MCC 12 GAG/gtaaga-> (SpliceGAG/gtaaaa Donor T16 MCC 145 ctcag/GGA-> (Splice atcag/GGA Acceptor T47MCC 267 CGG->CTG Arg->Leg T81 MCC 490 TCG->TTG Ser->Leu T35 MCC 506CGG->CAG Arg->Gln T91 MCC 698 GCT->GTT Ala->Val T34 APC 288CCAGT->CCCAGCCAGT (Insertion) T27 APC 331 CGA->TGA Arg->Stop T135 APC437 CAA/gtaa->CAA/gcaa (Splice Donor) T20I APC 1338 CAG->TAG Gln->StopFor splice site mutations, the codon nearest to the mutation is listedThe underlined nucleotide were mutated; small case letters representintrons, large case letters represent exonsFour of the mutations resulted in amino acid substitutions and tworesulted in the alteration of splice site consensus elements. Mutationsat analogous splice site positions in other genes have been shown toalter RNA processing in vivo and in vitro.

Three exons of APC were also evaluated in sporadic tumors. Sixty tumorswere screened by RNase protection, and an additional 98 tumors wereevaluated by sequencing. The exons examined included nt 822-930,931-1309, and 1406-1545 (Table I). A total of three mutations wereidentified, each of which proved to be somatic. Tumor T27 contained asomatic mutation of CGA (arginine) to TGA (stop codon) at codon 33.Tumor T135 contained a GT to GC change at a splice donor site. Tumor T34contained a 5 bp insertion (CAGCC between codons 288 and 289) resultingin a stop at codon 291 due to a frameshift.

We serendipitously discovered one additional somatic mutation in acolorectal cancer. During our attempt to define the sequences and splicepatterns of the MCC and APC gene products in colorectal epithelialcells, we cloned cDNA from the colorectal cancer cell line SW480. Theamino acid sequence of the MCC gene from SW480 was identical to thatpreviously found in clones from human brain. The sequence of APC inSW480 cells, however, differed significantly, in that a transition atcodon 1338 resulted in a change from glutamine (CAG) to a stop codes(TAG). To determine if this mutation was somatic, we recovered DNA fromarchival paraffin blocks of the original surgical specimen (T201) fromwhich the tumor cell line was derived 28 years ago.

DNA was purified from paraffin sections as described in S. E. Goelz, S.R. Hamilton, and B. Vogelstein. Biochem. Biophys. Res. Comm. 130, 118(1985). PCR was performed as described in reference 24, using theprimers 5′-GTTCCAGCAGTGTCACAG-3′ (SEQ ID NO:101) and5′-GGGAGATTTCGCTCCTGA-3′ (SEQ ID NO:102). A PCR product containing codon1338 was amplified from the archival DNA and used to show that the stopcodon represented a somatic mutation present in the original primarytumor and in cell lines derived from the primary and metastic tumorsites, but not from normal tissue of the patient.

The ten point mutation in the MCC and APC genes so far discovered insporadic. CRCs are summarized in Table IIB. Analysis of the number ofmutant and wild-type PCR clones obtained from each of these tumorsshowed that in eight of the ten cases, the wild-type sequence waspresent in approximately equal proportions to the mutant. This wasconfirmed by RFLP analysis using flanking markers from chromosome 5q wasdemonstrated that only two of the ten tumors (T135 and T201) exhibitedan allelic deletion on chromosome 5q. These results are consistent withprevious observations showing that 20-40% of sporadic colorectal tumorshad allelic deletions of chromosome 5q. Moreover, these data suggestthat mutations of 5q21 genes are not limited to those colorectal tumorswhich contain allelic deletions of this chromosome.

EXAMPLE 4

This example characterizes small, nested deletions in DNA from twounrelated FAP patients.

DNA from 40 FAP patients was screened with cosmids that has been mappedinto a region near the APC locus to identify small deletions orrearrangements. Two of these cosmids, L5.71=nd L5.79, hybridized with a1200 kb NotI fragment in DNAs from most of the FAP patients screened.

The DNA of one FAP patient, 3214 showed only a 940 kb NotI fragmentinstead of the expected 1200 kb fragment. DNA was analyzed from fourother members of the patient's immediate family; the 940 kb fragment waspresent in her affected mother (4711), but not in the other, unaffectedfamily members. The mother also carried a normal 1200 kb Not1 fragmentthat was transmitted to her two unaffected offspring. These observationsindicated that the mutant polyposis allele is on the same chromosome asthe 940 kb Not1 fragment. A simple interpretation is that APC patients3214 and 4711 each carry a 260 kb deletion within the APC locus.

If a deletion were present, then other enzymes might also be expected toproduce fragments with altered mobilities. Hybridization of L5.79 toNruI-digested DNAs from both affected members of the family revealed anovel NruI fragment of 1300 kb, in addition to the normal 1200 kb NruIfragment. Furthermore, M1u1 fragments in patients 3214 and 4711 alsoshowed an increase in size consistent with the deletion of an M1uI site.The two chromosome 5 homologs of patient 3214 were segregated in somaticcell hybrid lines; HHW1155 (deletion hybrid) carried the abnormalhomolog and HHW1159 (normal hybrid) carried the normal homolog.

Because patient 8214 showed bray only a 940 kb NotI fragment, she hadnot inherited the 1200 kb fragment present in the unaffected father'sDNA. This observation suggests that he must be heterozygous for, andhave transmitted, either a decision of the L5.79 probe region or avariant NotI fragment too large to resolve on the gel system. Asexpected, the hybrid cell line HHW1159, which carries the paternalhomolog, revealed no resolved Not fragment when probed with L5.79.However, probing of HHW1159 DNA with L5.79 following digestion withother enzymes did reveal restriction fragments, demonstrating thepresence of DNA homologous to the probe. The father is, therefore,interpreted as heterozygous for a polymorphism at the NotI site, withone chromosome 5 having a 1200 kb NotI fragment and the other having afragment too large to resolve consistently on the gel. The latter wastransmitted to patient 3214.

When double digests were used to order restriction sites within the 1200kb NotI fragment, L5.71 and L5.79 were beth both found to lie on a 550kb NotI-NruI fragment and, therefore, on the same side of an NruI sitein the 1200 kb NotI fragment. To obtain genomic representation ofsequences present over the entire 1200 kb NotI fragment, we constructeda library of small-fragment inserts enriched for sequences from thisfragment. DNA from the somatic cell hybrid HHW141, which contains about40% of chromosome 5, was digested with NotI and electrophoresed underpulsed-field gel (PPG) conditions; EcoRI fragments from the 1200 kbregion of this gel were cloned into a phage vector. Probe Map30 wasisolated from this library. In normal individuals probe Map30 hybridizesto the 1200 kb NotI fragment and to a 200 kb NruI fragment. This latterhybridization places Map30 distal, with respect to the locations ofL5.71 and L5.79, to the NruI site of the 550 kb NotI-NruI fragment.

Because Map30 hybridized to the abnormal, 1300 kb Nru1 fragment ofpatient 3214, the locus defined by Map30 lies outside the hypothesizeddeletion. Furthermore, in normal chromosomes Map30 identified a 200 kbNruI fragment and L5.79 identified a 1200 kb NruI fragment; thehypothesized deletion must, therefore, be removing an NruI site, orsites, lying between Map30 and L5.79, and these two probes must flankthe hypothesized deletion. A restriction map of the genomic region,showing placement of these probes, is shown in FIG. 5.

A NotI digest of DNA from another FAP patient, 3824, was probed withL5.79. In addition to the 1200 kb normal NotI fragment, a fragment ofapproximately 1100 kb was observed, consistent with the presence of a100 kb deletion in one chromosome 5. In this case, however, digestionwith NruI and M1uI did not reveal abnormal bands, indicating that if adeletion were present, its boundaries must lie distal to the NruI andM1uI sites of the fragments identified by L5.79. Consistent with thisexpectation, hybridization of Map30 to DNA from patient 3824 identifieda 760 kb M1uI fragment in addition to the expected 860 kb fragment,supporting the interpretation of a 100 kb deletion in this patient. Thetwo chromosome 5 homologs of patient 3824 were segregated in somaticcell hybrid lines; HHW1291 was found to carry only the abnormal homologand HHW1290 only the normal homolog.

That the 860 kb M1u1 fragment identified by Map30 is distinct from the830 kb M1uI fragment identified previously by L5.79 was demonstrated byhybridization of Map30 and L5.79 to a NotI-M1uI double digest of DNAfrom the hybrid cell (HHWW1159) containing the nondeleted chromosome 5homolog of patient 3214. As previously indicated, this hybrid isinterpreted as missing one of the NotI sites that define the 1200 kbfragment. A 620 kb NotI-M1uI fragment was seen with probe L5.79, and an860 kb fragment was seen with Map30. Therefore, the 830 kb M1uI fragmentrecognized by probe L5.79 must contain a NotI site in HHW1159 DNA;because the 800 kb M1uI fragment remains intact, it does not carry thisNotI site and must be distinct from the 830 kb M1u1 fragment.

EXAMPLE 5

This example demonstrates the isolation of human sequences which spanthe region deleted in the two unrelated FAP patients characterized inExample 4.

A strong prediction of the hybridization that patients 8214 and 3824carry deletions is that some sequences present on normal chromosome 5homologs would be missing from the hypothesized deletion homologs.Therefore, to develop genomic probes that might confirm the deletions,as well as to identify genes from the region, YAC clones from a contigseeded by cosmid L5.79 were localized from a library containing sevenhaploid human genome equivalents (Albertsen et al., Proc. Natl. Acad.Sci. U.S.A., Vol. 87, pp. 4256-4260 (1990)) with respect to thehypothesized deletions. Three clones, YACs 57B8, 310D8, and 183H12, werefound to overlap the deleted regions.

Importantly, one end of YAC 57B8 (clone AT57) was found to lie withinthe patient 3214 deletion. Inverse polymerase chain reaction (PCR)defined the end sequences of the insert of YAC 57b8. PCR primers basedon one of these end sequences repeatedly failed to amplify DNA from thesomatic cell hybrid (HHW1155) carrying the deleted homolog of patient3214, but did amplify a product of the expected size from the somaticcell hybrid (HHWW1159) carrying the normal chromosome 5 homolog. Thisresult support the interpretation that the abnormal restrictionfragments found in the DNA of patient 3214 result from a deletion.

Additional support for the hypothesis of deletions in DNA from patient3214 came from subcloned fragments of YAC 183H12, which spans the regionin question. Y11, an EcoRI fragment cloned from YAC 183H12, hybridizedto the normal, 1200 kb NotI fragment of patient 4711, but failed tohybridize to the abnormal, 940 kb Notl fragment of 4711 or to DNA fromdeletion cell line HHW1155. This result confirmed the deletion inpatient 3214.

Two additional EcoR1 fragments from YAC 183H12. Y10 and Y14, werelocalized within the patient 3214 deletion by their failure to hybridizeto DNA from HHW1155. Probe Y10 hybridizes to a 150 kb NruI fragment innormal chromosome 5 homologs. Because the 3214 deletion creates the 1300kb NruI fragment seen with the probes L5.79 and Map30 that flank thedeletion, these NruI sites and the 150 kb NruI fragment lying betweenmust be deleted in patient 3214. Furthermore, probe Y10 hybridizes tothe same 620 kb NotI-M1uI fragment seen with probe L5.79 in normal DNA,indicating its location as L5.79-proximal to the deleted M1uI site andplacing it between the M1uI site and the L5.79-proximal NruI site. TheM1uI site must, therefore, lie between the NruI sites that define the150 kb NruI fragment (see FIG. 5).

Probe Y11 also hybridized to the 150 kb NruI fragment in the normalchromosome 5 homolog, but failed to hybridize to the 620 Kb Not1-M1uIfragment, placing it L5.79-distal to the M1uI site, but proximal to thesecond NruI site. Hybridization to the same (860 kb) M1uI fragment asMap30 confirmed the localization of probe Y11 L5.79-distal to the M1uIsite.

Probe Y14 was shown to be L5.79-distal to both deleted NruI sites byvirtue of its hybridization to the same 200 kb NruI fragment of thenormal chromosome 5 seen with Map30. Therefore, the order of these EcoRIfragments derived from YAC 183H12 and deleted in patient 3214, withrespect to L5.79 and Map30, is L5.79-Y10-Y11-Y14-Map30.

The 100 kb deletion of patient 3284 was confirmed by the failure ofaberrant restriction fragments in this DNA to hybridize with probe Y11,combined with positive hybridizations to probes Y10 and/or Y14. Y10 andY14 each hybridized to the 1100 kb NotI fragment of patient 3824 as wellas to the normal 1200 kb NotI fragment, but Y11 hybridized to the 1200kb fragment only. In the M1u1 digest, probe Y14 hybridized to the 860 kband 760 kb fragments of patient 3824 DNA, but probe Y11 hybridized onlyto the 860 k13 fragment. We conclude that the basis for the alterationin fragment size in DNA from patient 3824 is, indeed, a deletion.Furthermore, because probes Y10 and Y14 are missing from the deleted3214 chromosome, but present on the deleted 3824 chromosome, and theyhave been shown to flank probe Y11, the deletion in patient 3824 must benested within the patient 3214 deletion.

Probes Y10, Y11, Y14 and Map30 each hybridized to YAC 310D8, indicatingthat this YAC spanned the patient 3824 deletion and at a minimum, mostof the 3214 deletion. The YAC characterizations, therefore, confirmedthe presence of deletions in the patients and provided physicalrepresentation of the deleted region.

EXAMPLE 6

This example demonstrates that the MCC coding sequence maps outside ofthe region deleted in the two FAP patients characterized in Example 4.

An intriguing FAP candidate gene, MCC, recently was ascertained withcosmid L5.71 and was shown to have undergone mutation in coloncarcinomas (Kinzler et al., supra). It was therefore of interest to mapthis gene with respect to the deletions in APC patients. Hybridizationof MCC probes with an overlapping series of YAC clones extending ineither direction from L5.71 showed that the 3′ end of MCC must beoriented toward the region of the two APC deletions.

Therefore, two 3′ cDNA clones from MCC were mapped with respect, to thedeletions: clone 1CI (bp 2378-4181) and clone 7 (bp 2890-3560). Clone1CI contains sequences from the C-terminal end of the open readingframe, which stops at nucleotide 2708, as well as 3′ untranslatedsequence. Clone 7 contains sequence that is entirely 3′ to the openreading frame. Importantly, the entire 3′ untranslated sequencecontained in the cDNA clones consists of a single 2.5 kb exon. These twoclones were hybridized to DNAs from the YACs spanning the FAP region.Clone 7 fails to hybridize to YAC 310D8, although it does hybridize toYACs 183H12 and 57B8; the same result was obtained with the cDNA 1CI.Furthermore, these probes did show hybridization to DNAs from bothhybrid cell lines (HWW1159 and HWW1155) and the lymphoblastoid cell linefrom patient 3214, confirming their locations outside the deletedregion. Additional mapping experiments suggested that the 3′ end of theMCC cDNA clone coding is likely to be located more than 45 kb from thedeletion of patient 3214 and, therefore, more than 100 kb from thedeletion of patient 3824.

EXAMPLE 7

This example identifies three genes within the deleted region ofchromosome 5 in two unrelated FAP patients characterized in Example 4.

Genomic clones were used to screen cDNA libraries in three separateexperiments. One screening was done with a phage clone derived from YAC310D8 known to span the 260 kb deletion of patient 3214. A large-insertphage library was constructed from this YAC; screening with Y11identified λ205, which mapped within both deletions. When clone λ205 wasused to probe a random-, plus oligo(dT)-, primed fetal brain cDNAlibrary (approximately 300,000 phage), six cDNA clones were isolated andeach of them mapped entirely within both deletions. Sequence analysis ofthese six clones formed a single cDNA contig, but did not reveal anextended open reading frame. One of the six cDNAs was used to isolatemore cDNA clones, some of which crossed the L5.71-proximal breakpoint ofthe 3824 deletion, as indicated by hybridization to both chromosome ofthis patient. These clones also contained an open reading frame,indicating a transcriptional orientation proximal to distal with respectto L5.71. This gene was named DP1 (deleted in polyposis 1). This gene isidentical to TB2 described above.

cDNA walks yielded a cDNA contig of 3.0-3.5 kb, and included two clonescontaining terminal poly(A) sequences. This size corresponds to the 3.5kb band seen by Northern analysis. Sequencing of the first 3163 bp ofthe cDNA contig revealed an open reading frame extending from the firstbase to nucleotide 631, followed by a 2.5 kb 3′ untranslated region. Thesequence surrounding the methionine codon at base 77 conforms to theKozak consensus of an initiation methionine (Kozak, 1984). Failedattempts to walk farther, coupled with the similarity of the lengths ofisolated cDNA and mRNA, suggested that the NH2-terminus of the DP1protein had been reached. Hybridization to a combination of genomic andYAC DNAs cut with various enzymes indicated the genomic coverage of DP1to be approximately 30 kb.

Two additional probes for the locus, YS-11 and YS-39, which had beenascertained by screening of a cDNA library with an independent YAC probeidentified with MCC sequences adjacent to L5.71, were mapped into thedeletion region. YS-39 was shown to be a cDNA identical in sequence toDP1. Partial characterization of YS-11 had shown that 200 bp of DNAsequence at one end was identical to sequence coding for the 19 kdprotein of the ribosomal signal recognition particle. SRP19 (Lingelbachet al., supra). Hybridization experiments mapped YS-11 within beth bothdeletions. The sequence of this clone, however, was found to be complex.Although 454 bp of the 1032 bp sequence of YS-11 were identical to theGenBank entry for the SRP19 gene, another 578 bp appended 5′ to theSRP19 sequence was found to consist of previously unreported sequencecontaining no extended open reading frames. This suggested that YS-11was either a chemeric clone containing two independent inserts or aclone of an incompletely processed or aberrant message. If YS-11 were aconventional chimeric clone, the independent segments would not beexpected to map to the same physical region. The segments resulting fromanomalous processing of a continuous transcript, however, would map to asingle chromosomal region.

Inverse PCR with primers specific to the two ends of YS-11, the SRP19,end and the unidentified region, verified that both sequences map withinthe YAC 310D8; therefore, YS-11 is most likely a clone of an immature oranomalous mRNA species. Subsequently, both ends were shown to lie withthe deleted region of patient 3824, and YS-11 was used to screen foradditional cDNA clones.

Of the 14 cDNA clones selected from the fetal brain library, one clone,V5, was of particular interest in that it contained an open readingframe throughout, although it included only a short identity to thefirst 78 5′ bases of the YS-11 sequence. Following the 78 bp ofidentical sequence, the two cDNA sequences diverged at an AG.Furthermore, divergence from genomic sequence was also seen after these78 bp, suggesting the presence of a splice junction, and supporting theview that YS-11 represents an irregular message.

Starting with V5, successive 5′ and 3′ walks were performed; theresulting cDNA coding consisted of more than 100 clones, which defined anew transcript, DP2. Clones walking in the 5′ direction crossed the 3824deletion breakpoint farthest from L5.71; since its 3′ end is closer tothis cosmid than its 5′ end, the transcriptional orientation of DP2 isopposite to that of MCC and DP1.

The third screening approach relied on hybridization with a 120 kb M1uIfragment with YAC 57B8. This fragment hybridizes with probe Y11 andcompletely spans the 100 kb deletion in patient 3824, the fragment waspurified on two preparative PFGs labeled, and used to screen a fetalbrain cDNA library. A number of cDNA clones previously identified in thedevelopment of the DP1 and DP2 contigs were reascertained. However, 19new cDNA clones mapped into the patient 3824 deletion. Analysisindicated that these 19 formed a new contig, DP3, containing a largeopen reading frame.

A clone from the 5′ end of this new cDNA contig hybridized to the sameEcoRI fragment as the 3′ end of DP2. Subsequently, the DP2 and DP3contigs were connected by a single 5′ walking step from DP3, to form thesingle contig DP2.5. The complete nucleotide sequence of DP2.5 is shownin FIG. 9.

The consensus cDNA sequence of DP2.5 suggests that the entire codingsequence of DP2.5 has been obtained and is 8532 bp long. The most 5′ ATGcodon occurs two codons from an in-frame stop and comforms to the Kozakinitiation consensus (Kozak, Nucl. Acids. Res., Vol. 12, p. 857-8721984). The 3′ open reading frame breaks down over the final 1.8 kb,giving multiple stops in all frames. A poly(A) sequence was found in oneclone approximately 1 kb into the 3′ untranslated region, associatedwith a polyadenylation signal 33 bp upstream (position 9530). The openreading frame is almost identical to the identified as APC above.

An alternatively spliced exon at nucleotide 934 of the DP2.5 transcriptof potential interest, it was first discovered by noting that twoclasses of cDNA had been isolated. The more abundant cDNA class containsa 303 bp exon not included in the other. The presence in vivo of the twotranscripts was verified by an exon connection experiment. Primersflanking the alternatively spliced exon were used to amplify, by PCR,cDNA prepared from various adult tissues. Two PCR products that differedin size by approximately 300 bases were amplified from all the tissuestested; the larger product was always more abundant than the smaller.

EXAMPLE 8

This example demonstrates the primers used to identify subtle mutationsin DP1, SRP19, and DP25.

To obtain DNA sequence adjacent to the exons of the genes DP1, DP2.5,and SRP19, sequencing substrate was obtained by inverse PCRamplification of DNAs from two YACs 310D8 and 183H12, that span thedeletions. Ligation at low concentration cyclized the restrictionenzyme-digested YAC DNAs. Oligonucleotides with sequencing tails,designed in inverse orientation at intervals along the cDNAs, primed PCRamplification form the cyclized templates. Comparison of these DNAsequences with the cDNA sequences placed exon boundaries at thedivergence points. SRP19 and DP1 were each shown to have five exons.DP2.5 consisted of 15 exons. The sequences of the oligonucleotidessynthesized to provide PCR amplification primers for the exons of eachof these genes are listed in Table III SEQ ID NO:39-94 .

TABLE III Sequence of Primers Used for SSCP Analyses Exon Primer 1Primer 2 DP1 UP-TCCCCGCCTGCCGCTCTC (SEQ ID NO:39) RP-GCAGCGGCGGCTCCCGTG(SEQ ID NO:40) UP-GTGAACGGCTCTCATGCTGC (SEQ ID NO:41)RP-ACGTGCGGGGAGGAATGGA (SEQ ID NO:42) UP-ATGATATCTTACCAAATGATATAC (SEQID NO:43) RP-TTATTCCTACTTCTTCTATACAG (SEQ ID NO:44)UP-TACCCATGCTGGCTCTTTTTC (SEQ ID NO:45) RP-TGGGGCCATCTTGTTCCTGA (SEQ IDNO:46) UP-ACATTAGGCACAAAGCTTGCAA (SEQ ID NO:47)RP-ATCAAGCTCCAGTAAGAAGGTA (SEQ ID NO:48) SRP19 UP-TGCGGCTCCTGGGTTGTTG(SEQ ID NO:49) RP-GCCCCTTCCTTTCTGAGGAC (SEQ ID NO:50)UP-TTTTCTCCTGCCTCTTACTGC (SEQ ID NO:51) RP-ATGACACCCCCCATTCCCTC (SEQ IDNO:52) UP-CCACTTAAAGCACATATATTTAGT (SEQ ID NO:53)RP-GTATGGAAAATAGTGAAGAACC (SEQ ID NO:54) UP-TTCTTAAGTCCTGTTTTTCTTTTG(SEQ ID NO:55) RP-TTTAGAACCTTTTTTGTGTTGTG (SEQ ID NO:56)UP-CTCAGATTATACACTAAGCCTAAC (SEQ ID NO:57) RP-CATGTCTCTTACAGTAGTACCA(SEQ ID NO:58) DP2.5 UP-AGGTCCAAGGGTAGCCAAGG* (SEQ ID NO:59)RP-TAAAAATGGATAAACTACAATTAAAAG (SEQ ID NO:60)UP-AAATACAGAATCATGTCTTGAAGT (SEQ ID NO:61) RP-ACACCTAAAGATGACAATTTGAG(SEQ ID NO:62) UP-TAACTTAGATAGCAGTAATTTCCC* (SEQ ID NO:63)RP-ACAATAAACTGGAGTACACAAGG (SEQ ID NO:64) UP-ATAGGTCATTGCTTCTTGCTGAT*(SEQ ID NO:65) RP-TGAATTTTAATGGATTACCTAGGT (SEQ ID NO:66)UP-CTTTTTTTGCTTTTACTGATTAACG (SEQ ID NO:67)RP-TGTAATTCATTTTATTCCTAATACCTC (SEQ ID NO:68)UP-GGTAGCCATAGTATGATTATTTCT (SEQ ID NO:69) RP-CTACCTATTTTTATACCCACAAAC(SEQ ID NO:70) UP-AAGAAAGCCTACACCATTTTTGC (SEQ ID NO:71)RP-GATCATTCTTAGAACCATCTTGC (SEQ ID NO:72) UP-ACCTATAGTCTAAATTATACCATC(SEQ ID NO:73) RP-GTCATGGCATTACTGACCAG (SEQ ID NO:74)UP-AGTCGTAATTTTGTTTCTAAACTC (SEQ ID NO:75) RP-TGAAGGACTCCGATTTCACCC*(SEQ ID NO:76) UP-TCATTCACTCACAGCCTGATGAC* (SEQ ID NO:77)RP-GCTTTGAAACATGCACTACGAT (SEQ ID NO:78) UP-AAACATCATTGCTCTTCAAATAAC(SEQ ID NO:79) RP-TACCATGATTTAAAAATCCACCAG (SEQ ID NO:80)UP-GATGATTGTCTTTTTCCTCTTTGC (SEQ ID NO:81) RP-CTGAGCTATCTTAAGAAATACATG(SEQ ID NO:82) UP-TTTTAAATGATCCTCTATTCTGTAT (SEQ ID NO:83)RP-ACAGAGTCAGACCCTCCCTCAAAG (SEQ ID NO:84) UP-TTTCATATTCTTACTGCTAGCATT(SEQ ID NO:85) RP-ATACACAGGTAAGAAATTAGGA (SEQ ID NO:86)UP-TAGATGACCCATATTCTCTTTC (SEQ ID NO:87) RP-CAATTAGGTCTTTTTGAGAGTA (SEQID NO:88) 3-A UP-GTTACTGCATACACATTGTGAC (SEQ ID NO.89)RP-GCTTTTTGTTTCGTAACATGAAG* (SEQ ID NO:90) B UP-AGTACAAGGATGCCAATATTATG*(SEQ ID NO:103) RP-ACTTCTATCTTTTTCAGAACGAG* (SEQ ID NO:104) CUP-ATTTGAATACTACAGTGTTACCC* (SEQ ID NO:105) RP-CTTGTATTCTAATTTGGCATAAGG*(SEQ ID NO:106) D UP-CTGCCCATACACATTCAAACAC* (SEQ ID NO:107)RP-TGTTTGCGTCTTGCCCATCTT* (SEQ ID NO:108) E UP-AGTCTTAAATATTCAGATGAGCAG*(SEQ ID NO:109) RP-GTTTCTCTTCATTATATTTTATGCTA* (SEQ ID NO:110) FUP-AAGCCTACCAATTATAGTGAACG* (SEQ ID NO:111) RP-AGCTGATGACAAAGATGATAATC*(SEQ ID NO:112) G UP-AAGAAACAATACAGACTTATTGTG* (SEQ ID NO:113)RP-ATGAGTGGGGTCTCCTGAAC* (SEQ ID NO:114) H UPATCTCCCTCCAAAAGTGGTGC* (SEQID NO:115) RP-TCCATCTGGAGTACTTTCTGTG* (SEQ ID NO:116) IUP-AGTAAATGCTGCAGTTCAGAGG* (SEQ ID NO:117) RP-CCGTGGCATATCATCCCCC* (SEQID NO:118) J UP-CCCAGACTGCTTCAAAATTACC* (SEQ ID NO:119)RP-GAGCCTCATCTGTACTTCTGC* (SEQ ID NO:120) K UP-CCCTCCAAATGAGTTAGCTGC*(SEQ ID NO:121) RP-TTGTGGTATAGGTTTTACTGGTG* (SEQ ID NO:122) LUP-ACCCAACAAAAATCAGTTAGATG* (SEQ ID NO:123) RP-GTGGCTGGTAACTTTAGCCTC*(SEQ ID NO:124) N UP-ATGATGTTGACCTTTCCAGGG* (SEQ ID NO:125)RP-ATTGTGTAACTTTTCATCAGTTGC* (SEQ ID NO:126) M UP-AAAGACATACCAGACAGAGGG*(SEQ ID NO:127) RP-CTTTTTTGGCATTGCGGAGCT* (SEQ ID NO:128) OUP-AAGATGACCTGTTGCAGGAATG* (SEQ ID NO:129) RP-GAATCAGACCAAGCTTGTCTAGAT*(SEQ ID NO:130) P UP-CAATAGTAAGTAGTTTACATCAAG* (SEQ ID NO:131)RP-AAACAGGACTTGTACTGTAGGA* (SEQ ID NQ:132) Q UP-CAGCCCCTTCAAGCAAACATC*(SEQ ID NO:133) RP-GAGGACTTATTCCATTTCTACC* (SEQ ID NO:134) RUP-CAGTCTCCTGGCCGAAACTC* (SEQ ID NO:135) RP-GTTGACTGGCGTACTAATACAG* (SEQID NO:136) S UP-TGGTAATGGAGCCAATAAAAAGG* (SEQ ID NO:137)RP-TGGGACTTTTCGCCATCCAC* (SEQ ID NO:138) T UP-TGTCTCTATCCACACATTCGTC*(SEQ ID NO:139) RP-ATGTTTTTCATCCTCACTTTTGC* (SEQ ID NO:140) UUP-GGAGAAGAACTGGAAGTTCATC* (SEQ ID NO:141) RP-TTGAATCTTTAATGTTTGGATTTGC*(SEQ ID NO:142) V UP-TCTCCCACAGGTAATACTCCC (SEQ ID NO:143)RP-GCTACAACTGAATGGGGTACG (SEQ ID NO:144) W UP-CAGGACAAAATAATCCTGTCCC(SEQ ID NO:145) RP-ATTTTCTTACTTTCATTCTTCCTC (SEQ ID NO:146) All primersare read in the 5′ to 3′ direction, the first primer in each pair lies5′ of the exon it amplifies: the second primer lies 3′ of the exon itamplifies. Primers that lie within the exon are identified by anasterisk. UP represents the 21M13 universal primer sequence[:]. RPrepresents the M13 reverse primer sequence.With the exception of exons 1, 3, 4, 9, and 15 of DP2.5 (see below), theprimer sequences were located in intron sequences flanking the exons.The 5′ primer of exon 1 is complementary to the cDNA sequence, butextends just into the 5′ Kozak consensus sequence for the initiatormethionine, allowing a survey of the translated sequences. The 5′ primerof exon 3 is actually in the 5′ coding sequences of this exon, as threeseparate intronic primers simply would not amplify. The 5′ primer ofexon 4 just overlaps the 5′ end of this exon, and we thus fail to surveythe 19 most 5′ bases of this exon. For exon 9, two overlapping primersets were used, such that each had one end within the exon. For exon 15,the large 3′ exon of DP2.5, overlapping primer pairs were placed alongthe length of the exon; each pair amplified a product of 250-400 bases.

EXAMPLE 9

This example demonstrates the use of single stranded conformationpolymorphism (SSCP) analysis as described by Orita et al. Proc. Natl.Acad. Sci. U.S.A., Vol. 86, pp. 2766-70 (1989) and Genomics, Vol. 5, pp.874-879 (1989) as applied to DP1, SRP19 and DP2.5.

SSCP analysis identifies most single- or multiple-base changes in DNAfragments up to 400 bases in length. Sequence alterations are detectedas shifts in electrophoretic mobility of single-stranded DNA onnondenaturing acrylamide gels; the two complementary strands of DNAsegment usually resolve as two SSCP conformers of distinct mobilities.However, if the sample is from an individual heterozygous for abase-pair variant within the amplified segment, often three or morebands are seen. In some cases, even the sample from a homozygousindividual will show multiple bands. Base-pair-change variants areidentified by differences in pattern among the DNAs of the sample set.

Exons of the candidate genes were amplified by PCR from the DNAs of 61unrelated FAP patients and a control set of 12 normal individuals. Thefive exons from DP1 revealed no unique conformers in the FAP patients,although common conformers were observed with exons 2 and 3 in someindividuals of both affected and control sets, indicating the presenceof DNA sequence polymorphisms. Likewise, none of the five exons of SRP19revealed unique conformers in DNA from FAP patients in the test panel.

Testing of exons 1 through 14 and primer sets A through N of exon 15, ofthe DP2.5 gene, however, revealed variant conformers specific to FAPpatients in exons 7, 8, 10, 11, and 15. These variants were in theunrelated patients 3746, 3460, 3827, 3712, and 3751, respectively. ThePCR-SSCP procedure was repeated for each of these exons in the fiveaffected individuals and in an expanded set of 48 normal controls. Thevariant bands were reproducible in the FAP patients but were notobserved in any of the control DNA samples. Additional variantconformers in exons 11 and 15 of the DP2.5 gene were seen; however, eachof these was found in both the affected and control DNA sets. The fivesets of conformers unique to the FAP patients were sequenced todetermine the nucleotide changes responsible for their alteredmobilities. The normal conformers from the host individuals weresequenced also. Bands were cut from the dried acrylamide gels, and theDNA was eluted. PCR amplification of these DNAs provided template forsequencing.

The sequences of the unique conformers from exons 7, 8, 10, and 11 ofDP2.5 revealed dramatic mutations in the DP2.5 gene. The sequence of thenew mutation creating the exon 7 conformer in patient 3746 was shown tocontain deletion of two adjacent nucleotides, at positions 730 and 731in the cDNA sequence (FIG. 7, SEQ ID NO:1). The normal sequence at thissplice junction is CAGGGTCA (intronic sequence underlined), with theintron-exon boundary between the two repetitions of AG. The mutantallele in this patient has the sequence CAGGTCA. Although this change isat the 5′ splice site, comparison with known consensus sequences ofsplice junctions would suggest that a functional splice junction ismaintained. If this new splice junction were functional, the mutationwould introduce a frameshift that creates a stop codon 15 nucleotidesdown-stream. If the new splice junction were not functional, messengerprocessing would be significantly altered.

To confirm the 2-base deletion, the PCR product from FAP patient 3746and a control DNA were electrophoresed on an acrylamide-urea denaturinggel, along with the products of a sequencing reaction. The sample frompatient 3746 showed two bands differing in size by 2 nucleotides, withthe larger band identical in mobility to the control sample; this resultwas independent confirmation that patient, 3746 is heterozygous for a 2bp deletion.

The unique conformer found in exon 8 of patient 3460 was found to carrya C-T transition, at position 904 in the cDNA sequence of DP2.5 (shownin FIG. 7) , which replaced the normal sequence of CGA with TGA. Thispoint mutation, when read in frame, results in a stop codon replacingthe normal arginine codon. This single-base change had occurred withinthe context of a CG dimer, a potential hot spot for mutation (Barker etal., 1984).

The conformer unique to FAP patient 3827 in exon 10 was found to containa deletion of one nucleoside (1367, 1368, or 1369) when compared to thenormal sequence found in the other bands on the SSCP gel. This deletion,occurring within a set of three T's, changed the sequence from CTTTCA toCTTCA; this 1 base frameshift creates a downstream stop within 30 bases.The PCR product amplified from this patient's DNA also waselectrophoresed on an acrylamide-urea denaturing gel, along with the PCRproduct from a control DNA and products from a sequencing reaction. Thepatient's PCR product showed two bands differing by 1 bp in length, withthe larger identical in mobility to the PCR product from the normal DNA;this result confirmed the presence of a 1 bp deletion in patient 3827.

Sequence analysis of the variant conformer of exon 11 from patient 3712revealed the substitution of a T by a G at position changing the normaltyrosine codon to a stop codon.

The pair of conformers observed in exon 15 of the DP2.5 gene for FAPpatient 3751 also was sequenced. These conformers were found to carry anucleotide substitution of C to G at position 5253, the third base of avaline codon. No amino acid change resulted from this substitution,suggesting that this conformer reflects a genetically silentpolymorphism.

The observation of distinct inactivating mutations in the DP2.5 gene infour unrelated patients strongly suggested that DP2.5 is the geneinvolved in FAP. These mutations are summarized in Table IIA.

EXAMPLE 10

This example demonstrates that the mutations identified in the DP2.5(APC) gene segregate with the FAP phenotype.

Patient 3746, described above as carrying an APC allele with aframeshift mutation, is an affected offspring of two normal parents.Colonoscopy revealed no polyps in either parent nor among the patient'sthree siblings.

DNA samples from both parents, from the patient's wife, and from theirthree children were examined. SSCP analysis of DNA from both of thepatients's patient's parents displayed the normal pattern of conformersfor exon 7, as did DNA from the patient's wife and one of thisoff-spring. The two other children, however, displayed the same newconformers as their affected father. Testing of the patient and hisparents with highly polymorphic VNTR (variable number of tandem repeat)markers showed a 99.98% likelihood that they are his biological parents.

These obserations confirmed that this novel conformer, known to reflecta 2 bp deletion mutation in the DP2.5 gene, appeared spontaneously withFAP in this pedigree and was transmitted to two of the children of theaffected individual.

EXAMPLE 11

This example demonstrates polymorphisms in the APC gene which appear tobe unrelated to disease (FAP).

Sequencing of variant conformers found among controls as well asindividuals with APC has revealed the following polymorphisms in the APCgene; first, in exon 11, at position 1458, a substitution of T to Ccreating an RsaI restriction site but no amino acid change; and second,in exon 15, at positions 5037 and 5271, substitutions of A To G and G toT, respectively, neither resulting in amino acid substitutions. Thesenucleotide polymorphisms in the APC gene sequence may be useful fordiagnostic purposes.

EXAMPLE 12

This example shows the structure of the APC gene.

The structure of the APC gene is schematically shown in FIG. 8, withflanking intron sequences indicated (SEQ ID NO:11-38).

The continuity of the very large (6.5 kb), most 3′ exon in DP2.5 wasshown in two ways. First, inverse PCR with primers spanning the entirelength of this exon revealed no divergence of the cDNA sequence from thegenomic sequence. Second, PCR amplification with converging primersplaced at intervals along the exon generated products of the same sizewhether amplified from the originally isolated cDNA, cDNA from varioustissues, or genomic template. Two forms of exon 9 were found in DP2.5;one is the complete exon; and the other, labeled exon 9A, is the resultof a splice into the interior of the exon that deletes bases 934 to 1236in the mRNA and removes 101 amino acids from the predicted protein (seeFIG. 3 FIGS. 3A-3Z, SEQ ID NO:1 and 2).

EXAMPLE 13

This example demonstrates the mapping of the FAP deletions with respectto the APC exons.

Somatic cell hybrids carrying the segregated chromosomes 5 from the 100kb (HHW1291) and 260 kb (HHW1155) deletion patients were used todetermine the distribution of the APC genes exons across the deletions.DNAs from these cell lines were used as template, along with genomic DNAfrom a normal control, for PCR-based amplification of the APC exons.

PCR analysis of the hybrids from the 260 kb deletion of patient 3214showed that all but one (exon 1) of the APC exons are removed by thisdeletion. PCR analysis of the somatic cell hybrid HHW1291, carrying thechromosome 5 homolog with the 100 kb deletion from patient 3824,revealed that exons 1 through 9 are present but exons 10 through 15 aremissing. This result placed the deletion breakpoint either between exons9 and 10 or within exon 10.

EXAMPLE 14

This example demonstrates the expression of alternately spliced APCmessenger in normal tissues and in cancer cell lines.

Tissues that express the APC gene were identified by PCR amplificationof cDNA made to mRNA with primers located within adjacent APC exons. Inaddition, PCR primers that flank the alternatively spliced exon 9 werechosen so that the expression pattern of both splice forms could beasserted. All tissue types tested (brain, lung, aorta, spleen, heart,kidney, liver, stomach, placenta, and colonic mucosa) and cultured celllines (lymphoblasts, HL60, and choriocarcinoma) expressed both spliceforms of the APC gene. We note, however, that expression by lymphocytesnormally residing in some tissues, including colon, prevents unequivocalassessment of expression. The large mRNA, containing the complete exon 9rather than only exon 9A, appears to be the more abundant message.

Northern analysis of poly(A)-selected RNA from lymphoblasts revealed asingle band of approximately 10 kb, consistently with the size of thesequenced cDNA.

EXAMPLE 15

This example discusses structural features of the APC protein predictedfrom the sequence.

The cDNA consensus sequence of APC predicts that the longer, moreabundant form of the message codes for a 2842 or 2844 2843 amino acidpeptide with a mass of 311.8 kd. This predicted APC peptide was comparedwith the current data bases of protein and DNA sequences using bothIntelligenetics and GCG software packages. No genes with a high degreeof amino acid sequence similarity were found. Although many short(approximately 20 amino arid) regions of sequence similarity wereuncovered, none was sufficiently strong to reveal which, if any, mightrepresent functional homology. Interestingly, multiple similarities tomyosins and keratins did appear. The APC gene also was scanned forsequence motifs of known function; although multiple glycosylation,phosphorylation, and myristoylation sites were seen, their significanceis uncertain.

Analysis of the APC peptide sequence did identify features important inconsidering potential protein structure. Hydropathy plots (Kyte andDoolittle, J. Mol. Biol. Vol. 157, pp. 105-132(1982)) indicate that theAPC protein is notably hydrophilic. No hydrophobic domains suggesting asignal peptide or a membrane-spanning domain were found. Analysis of thefirst 1000 residues indicates that α-helical rods may form (Cohen andParry, Trends Biochem, Sci. Vol. 77, pp. 245-248 (1986); there is ascarcity of proline residues and, there are a number of regionscontaining heptad repeats (apolar-X-X-apolar-X-X-X). Interestingly, inexon 9A, the deleted form of exon 9, two heptad repeat regions arereconnected in the proper heptad repeat frame, deleting the interveningpeptide region. After the first 1000 residues, the high proline contentof the remainder of the peptide suggests a compact rather than arod-like structure.

The most prominent feature of the second 1000 residues is a 20 aminoacid repeat that is iterated seven times with semiregular spacing (Table4). The intervening sequences between the seven repeat regions contained114, 116, 151, 205, 107, and 58 amino acids, respectively. Finally,residues 2200-24000 contain a 200 amino acid basic domain.

TABLE IV Seven Different Versions of the 20-Amino Acid Repeat Consensus:F * V E * T P * C F S R * S S L S S L S (SEQ ID NO:147) 1262: Y C V E DT P I C F S R C S S L S S L S (SEQ ID NO:148) 1376: H T V Q E T P L M FS R C T S V S S L D (SEQ ID NO:149) 1492: F A T E S T P D G F S C S S SL S A L S (SEQ ID NO:150) 1643: Y C V E G T P I N F S T A T S L S D L T(SEQ ID NO:151) 1848: T P I E G T P Y C F S R N D S L S S L D (SEQ IDNO:152) 1953: F A I E N T P V C P S H N S S L S S L S (SEQ ID NO:153)2013: R H V E D T P V C F S R N S S L S S L S (SEQ ID NO:154) Numbersdenote the first amino acid of each repeat. The consensus sequence atthe top reflects a majority amino acid at a given position. In theconsensus sequence. “*” indicates “Xaa”

1. A preparation of antibodies which specifically binds to a human APC(adenomatous polyposis coli) protein having an amino acid sequence asshown in SEQ ID NO:1, 2, or 7, SEQ ID NO:2 or 7 and does notspecifically bind to other human proteins.
 2. A preparation ofantibodies which specifically binds to a human APC protein which is theproduct of a mutant allele found in tumor, wherein the antibodies do notspecifically bind to other human proteins, and wherein the human APCprotein is a mutant form of the amino acid sequence shown in SEQ IDNOS:2 and 7, and the mutant allele is a mutant form of the nucleotidesequence shown in SEQ ID NO:1.
 3. The preparation of claim 2 wherein themutant allele contains a mutation selected from the group consisting ofmutations at codons 243, 279, 288, 301,331,413,437, 456, 500, 712, and1338.
 4. The preparation of claim 2 wherein the mutant allele contains apremature stop codon.
 5. The preparation of claim 2 wherein the mutantallele contains a missense mutation.
 6. The preparation of claim 2wherein the mutant allele contains a frameshift mutation.
 7. Thepreparation of claim 2 wherein the mutant allele contains a splicejunction mutation.
 8. The preparation of claim 2 wherein the mutantallele contains an insertion mutation.